Infection with high oncogenic risk human papillomavirus types is the etiological factor of cervical cancer and a major cause of other epithelial malignancies, including vulvar, vaginal, anal, penile and head and neck carcinomas. These agents affect epithelial homeostasis through the expression of specific proteins that deregulate important cellular signaling pathways to achieve efficient viral replication. Among the major targets of viral proteins are components of the DNA damage detection and repair machinery. The activation of many of these cellular factors is critical to process viral genome replication intermediates and, consequently, to sustain faithful viral progeny production. In addition to the important role of cellular DNA repair machinery in the infective human papillomavirus cycle, alterations in the expression and activity of many of its components are observed in human papillomavirus-related tumors. Several studies from different laboratories have reported the impact of the expression of human papillomavirus oncogenes, mainly E6 and E7, on proteins in almost all the main cellular DNA repair mechanisms. This has direct consequences on cellular transformation since it causes the accumulation of point mutations, insertions and deletions of short nucleotide stretches, as well as numerical and structural chromosomal alterations characteristic of tumor cells. On the other hand, it is clear that human papillomavirus-transformed cells depend on the preservation of a basal cellular DNA repair activity level to maintain tumor cell viability. In this review, we summarize the data concerning the effect of human papillomavirus infection on DNA repair mechanisms. In addition, we discuss the potential of exploiting human papillomavirus-transformed cell dependency on DNA repair pathways as effective antitumoral therapies. Papillomaviridae family and, to date, over 200 different types have been identified and their genomes completely sequenced Human papillomaviruses (HPVs) are small, nonenveloped viruses with a genome composed of double-stranded circular DNA of approximately 8 kbp Approximately 40 HPV types infect the mucosa and skin of the anogenital tract. These HPV types are further subclassified as low or high risk according to their association with the development of cervical carcinoma and its precursor lesions. Low-risk HPV types, such as HPV6 and HPV11, are associated with genital warts and low-grade cervical intraepithelial neoplasia (CIN) To establish a productive infection, HPVs have to reach cells from the basal layer of the epithelium. These are the only keratinocytes of the tissue that proliferate and express the DNA replication machinery. Therefore, they provide a permissive environment for initial viral genome replication. The virus can reach these cells through microwounds caused during sexual intercourse or at specific anatomical sites were the basal layer is readily accessible, such as the squamocolumnar junction (SCJ) of the cervix In high-grade CIN and HPV-associated tumors, viral DNA is frequently integrated into the host cell genome. Viral integration is an accidental event that usually disrupts the region were the E1 and E2 genes are located, leading to the loss of their expression. This constitutes a critical step in HPV-associated carcinogenesis since the loss of E2 leads to the upregulated expression of the main viral oncoproteins, namely, E6 and E7 It is estimated that approximately 291 million women are infected by genital HPV types worldwide. Each year, nearly 500,000 new cases of cervical cancer are diagnosed and over 270,000 women die from this disease around the world. Importantly, most of the cases (∼86%) and deaths (88%) occur in developing countries DNA contains the essential information for the formation and function of an organism, and its preservation is critical for survival. DNA is a fairly stable macromolecule; however, it suffers constant attacks from extracellular and intracellular factors that jeopardize its integrity. These factors include chemical and physical agents, infectious agents, highly reactive metabolic byproducts, and replication errors DNA repair is the process by which alterations in DNA are reverted. Usually, this involves the action of nucleases, helicases, ligases and DNA polymerases. Different specialized DNA repair mechanisms exist in which complex arrays of cellular proteins detect and repair different kinds of DNA lesions The DNA damage response (DDR) is composed of different signal transduction pathways that include damage sensors, signal transducers and effectors that act in a coordinated fashion to maintain genome integrity. Some DNA lesions activate specific sensors, such as the MRN complex (Mre11-RAD50-NSB1), replication protein A (RPA) or ATR-interacting protein (ATRIP), that trigger signaling pathways by the phosphorylation of apical protein kinases, such as ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR) and DNA-dependent protein kinase (DNA-PK). Different types of DNA lesions activate different kinases Many viruses, especially those with oncogenic properties, express proteins that affect the cell cycle and DNA damage repair regulatory pathways. Several studies have shown that during genome amplification, HPV proteins interact with different components of the cellular DNA repair machinery to activate or downregulate the expression or activity of factors of the ATM and ATR pathways. In 2009, Moody and coworkers showed that ATM activation is required for the productive genomic replication of HPV31 but not for episomal maintenance Importantly, many proteins from ATM and ATR pathways colocalize with HPV replication sites, further supporting the role of these factors in the HPV life cycle. Different studies have shown that E1 and E2 early proteins may activate the DDR, recruiting factors involved in DNA repair to process replication intermediates during viral genome amplification. Identified proteins at centers of HPV replication include 53BP1, ATRIP, OPbp1, p-ATM, p-H2AX, p-p53, ATR, CHK1, CHK2, PCNA, RPA, NBS1, BRCA1, RAD51, MRE11 and Ku70/80 The effect of specific HPV proteins on DNA repair pathways has been addressed by different groups. The HPV genome does not express proteins capable of mediating viral DNA replication and depends on the host cell machinery to achieve genome amplification. Therefore, it must induce the host cell to re-enter the S phase of the cell cycle to obtain access to the DNA replication machinery p110, p107 and p130 (pRb2). This interrupts the interaction of pRb proteins with members of the E2F transcription factor family. Once free from inhibition, E2F activates the transcription of several genes involved in S phase induction and progression, including cyclins A and E Among the main targets of E6 is the tumor suppressor protein p53. E6 forms a ternary complex with E6-associated protein (E6AP) and p53, altering p53's functional capacity and inducing the degradation of this cellular protein by the ubiquitin-mediated proteolysis pathway Due to their impact on different cellular pathways, HPV oncoproteins may also promote the accumulation of genomic alterations that contribute to malignant transformation Several studies have shown that ATM and ATR pathways are targeted by HPV oncoproteins. For instance, Banerjee et al. Other alterations in DNA repair systems associated with HPVs have been described by different groups. One report has shown that oral keratinocytes immortalized with HPV16 exhibit deficiencies in the NER system and, consequently, are unable to remove cyclobutane pyrimidine dimers (CPDs) induced by UV light 6-methylguanine DNA methyltransferase (MGMT) degradation via the ubiquitin/proteasome pathway in a process that requires the interaction of E6 with E6AP. The action of MGMT, which is impaired by E6, protects cells and tissues against the effects of alkylating agents The oncoprotein E6 also targets DNA repair pathways. For instance, epithelial mammary cells expressing this protein from HPV16 exhibit a reduced capacity for removing thymine dimers after exposure to UV light These observations show that HPV infection may increase the frequency of DNA alterations in the host cell and delay the removal of these alterations by targeting DNA repair pathways. In addition, by impairing cell cycle checkpoints and apoptosis, HPV oncoproteins cause sustained proliferation while preventing cell death. Collectively, these effects may lead to cellular alterations that give rise to precursor lesions with a tendency for malignant progression.During tumor evolution, cells acquire genetic alterations that may upregulate the expression of proto-oncogenes or induce gain-of-function mutations in these genes. In addition, mutations, deletions and alterations in the methylation pattern of promoter sequences may downregulate the activity of tumor suppressor genes. Cells harboring these alterations may have proliferative advantages and acquire other phenotypical alterations, such as the capacity to invade other tissues and organs In vitro data indicate that the activation of this pathway is mediated by E7 and is characterized by the formation of large FANCD2 foci and the recruitment of FANCD2 and FANCD1/BRCA2 to chromatin In hereditary cancers, mutations transmitted from progenitors predispose the host to the development of certain types of tumors and/or increase their sensitivity to carcinogens. This process may involve alterations in components of different DNA repair pathways. Several studies have suggested that mutations in this group of genes may act as cofactors in the establishment and progression of HPV-associated tumors. For instance, patients with Fanconi anemia (FA) or individuals carrying mutations in the breast cancer-associated (BRCA) gene exhibit a higher risk of developing HPV-associated cancerous lesions XRCC1, ERCC2, ERCC4 and ERCC1, at both the mRNA and protein levels, was downregulated in tumors and precursor lesions compared to samples from control subjects Alterations in the expression of genes involved in DNA damage sensing and repair are readily detected in HPV-associated cancers and precursor lesions. A study that analyzed the expression of genes involved in BER in 50 invasive cervical cancer patients and 40 squamous intraepithelial lesions (SILs) showed that the expression of Genome instability, in the context of HPV infection, may arise through different molecular pathways. A clear example of this is represented by the results of a study conducted by Kadaja et al. Chronic inflammation also plays a major role in cancer biology As previously stated, HPV oncogenes E6 and E7 play a critical role in HPV-mediated carcinogenesis by affecting major cellular processes. The consequences of HPV oncoprotein actions are reflected on cellular genome homeostasis. For instance, inhibition of the postmitotic checkpoint by high-risk HPV E6 and E7 is associated with the induction of polyploidy in human keratinocytes Alterations in the regulation of the cell cycle and the inhibition of proapoptotic factors are important underlying events in HPV-mediated genome instability. As expected, genomic instability is an early event during HPV infection that precedes viral integration, the development of associated lesions and their eventual progression to cancer Genome instability is probably not a consequence of HPV-mediated malignant transformation. However, it may favor the acquisition of genetic alterations that confer growing advantages to cells expressing viral oncogenes. In addition, the underlying mutator phenotype may allow transformed cells to adapt rapidly to the harsh conditions of the tumor microenvironment, contributing to tumor onset and progression.The data discussed above further support the established notion that the main mechanism by which HPV induces cell transformation is the targeting of p53 and pRb by E6 and E7, respectively However, the implementation of new therapeutic strategies to treat HPV-associated tumors is still required. In this context, the dependence of HPV-transformed cells on cellular DNA repair machinery may constitute a suitable target for intervention. In fact, inhibitors of selected molecules involved in specific cellular pathways have been applied for the treatment of cervical cancer in clinical trials The first case of synthetic lethality involving genes associated with DNA damage repair was with poly(ADP-ribose) polymerase (PARP) and breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 E6 and E7 form high-risk HPV types are pleotropic proteins that target an increasing list of cellular factors affecting their expression and function. As such, HPV-transformed cells exhibit major alterations in important signaling pathways and cellular processes, including several DNA damage repair mechanisms. This fact has consequences of clinical relevance. For instance, patients harboring HPV-associated head and neck squamous cell carcinoma (HNSCC) have significantly improved survival compared with patients affected by HPV-negative tumors. Although the molecular mechanisms underlying this difference are not completely understood, it is accepted that impaired DNA repair abilities, probably due to HPV oncoproteins action, play a major role Prati B prepared the text describing the effect of HPV on DNA repair machinery. Marangoni B prepared the text describing HPV-associated disease treatment strategies. Boccardo E prepared, revised and corrected all the text.


Human embryonic stem cells (hESCs) have the potential to give rise to all types of cells in the humanbody when appropriately induced to differentiate. Stem cells can differentiate spontaneously into the three-germ layerderivatives by embryoid bodies (EBs) formation. However, the two-dimensional (2D) adherent culture of hESCs underdefined conditions is commonly used for directed differentiation toward a specific type of mature cells. In this study, weaimed to determine the propensity of the Royan hESC lines based on comparison of expression levels of 46 lineagespecific markers. In this experimental study, we have compared the expression of lineage-specific markers inhESC lines during EB versus adherent-based spontaneous differentiation. We used quantitative real-time polymerasechain reaction (qRT-PCR) to assess expressions of 46 lineage-specific markers in 4 hESC lines, Royan H1 (RH1),RH2, RH5, and RH6, during spontaneous differentiation in both EB and adherent cultures at 0, 10, and 30 days afterinitiation of differentiation. Based on qRT-PCR data analysis, the liver and neuronal markers had higher expression levels in EBs,whereas skin-specific markers expressed at higher levels in the adherent culture. The results showed differentialexpression patterns of some lineage-specific markers in EBs compared with the adherent cultures.According to these results, possibly the spontaneous differentiation technique could be a useful methodfor optimization of culture conditions to differentiate stem cells into specific cell types such ectoderm, neuron, endodermand hepatocyte. This approach might prove beneficial for further work on maximizing the efficiency of directeddifferentiation and development of novel differentiation protocols. Embryonic stem cells (ESCs) divide indefinitely and give rise to all different cell types during differentiation. These unique properties make them invaluable cell sources for a wide range of applications in regenerative medicine, cell therapy, disease modeling, drug screening, gene delivery, and other research. However, despite their potential, human ESCs (hESCs) have not been successfully introduced into the clinic setting. Numerous obstacles must be overcome prior to their efficient use in cell replacement therapies. In addition to ethical issues that concern the embryonic source of hESCs , the moOptimization of the current differentiation protocols is a major challenge in stem cell research. The ability to efficiently generate pure populations of mature cell types from stem cells over a reasonable period of time is a major challenge. There are several protocols that enable differentiation of hESCs into diverse cell types like cardiomyocytes , 7, dopaThe following 3 main methods are commonly used for stem cell culture: i. Suspension culture and formation of embryoid bodies (EB), ii. Monolayer culture of stem cells on extracellular matrix proteins, and iii. Culture of ESCs on a feeder layer . Some pSome of the directed differentiation protocols include an initial step of EB mediated spontaneous differentiation followed by addition of soluble inducing factors to the cell aggregates. The differences in dimensions of the suspension and adherent culture systems result in significant differences between cell-cell and cell-matrix interactions, which highlights the importance of a proper choice of a 2D versus 3D stem cell culture system. Several reports have theoretically compared the adherent and suspension culture systems and their effects on the overall differentiation efficiency of stem cells, but there is no report of an experimental comparison , 27. In Different signal transduction pathways play rolesin the in vivo differentiation of stem cells into eachtype of mature cells. The microenvironmental factors that govern a cell’s fate in the body include structural, biochemical, mechanical, physiological, and hydrostatic stimuli . The envEB formation and spontaneous differentiation of ESCs result in heterogeneous populations of differentiated cells that include cells of the primary germ layers as well as some of the mature cell types that they produce. Spontaneous differentiation could be the first step for production of pure populations of desired cell types. The convenience of spontaneously differentiated ESCs makes them a useful research tool to provide researchers with valuable information about stem cell behavior, even during directed differentiation. It has been shown that comparative expression of lineage-specific markers during spontaneous differentiation of ESC lines is a good representative of their relative potentials for directed differentiation toward different lineages , 30. In In this study, we used spontaneous differentiation to optimize differentiation in 4 Royan hESC lines. These hESC lines were cultured under both adherent and suspension culture conditions and we compared expression levels of 46 lineage specific markers to determine the propensity of each of the Royan hESC lines. Subsequently, direct differentiation for neural cells and hepatocytes was done to confirm the spontaneousdifferentiation results.We evaluated our hypothesis by focusing on the expression level of neural stem cells lineage specific markers (in two undifferentiated and differentiated states with adherent or suspension culture system) like NESTIN, SOX1, NEUROD1, NCAM, PAX6, PDGFRa and GFAP , and b-Tubulin   in spontIn the other side we evaluated hepatocytedifferentiation potency by focusing on spontaneous anddirect endodermal layer differentiation and checking the general hepatic lineage specific expression markers like BRACHYURY, GOOSECOID, and SOX17 in two different culturing strategies (adherent and suspension). In this experimental study, approved by the Ethical Committee of Royan Institute, we have used 4 hESC lines-Royan H1 (RH1), RH2, RH5, and RH6 . RH6 isIn order to form EBs, we cultured the hESCs insuspension stem cell medium that contained agarose andfetal bovine serum , without KOSR. The samples were harvested at days 10 and 30 after the initiation of EB formation. For spontaneous differentiation in the adherent settings, we used the same media with slight changes. The cells were cultured on 0.1% gelatincoated plates instead of agarose coated plates.Total RNA was extracted using TRIzol  according to the manufacturer’s protocol. The RNAconcentration was measured using a Biowave WPA (S 2100) spectrophotometer. We examined the purity of each RNA sample based on 260/280 absorbance ratios. We noted that all samples had a ratio of 1.9-2. The quality of total RNA from all samples was determined by electrophoresis. Ribosomal 50S and 28S bands were sharp and showed approximately 2:1 band intensity, which confirmed that the samples had acceptable RNAintegrity and quality.We used a DNase treatment kit  based on the manufacturer’s instructions for elimination of DNA contamination. Random hexamer primers were used for first strand cDNA synthesis using the Fermentas kit. GAPDH and ß-Actin, as they have the most homogenous expression level among the Royan hESC lines based on the NormFinder software analysis (data not shown). The geometric mean Ct of GAPDH and ß-Actin were calculated for each sample, and the Ct results from all 46 genes were normalized based on the mean housekeeping values.Totally, we used 48 primers in this study. The sequences of 34 primers were obtained from  and 13 RH5 hESCs were differentiated to neuronal cells using 2 different protocols for confirmation of the spontaneous differentiation results. These protocols consisted of 3 main steps: i. Induction of hESC colonies toward neural ectoderm, ii. Differentiation toward neural tube formation, and iii. Neuron maturation stage . Steps iWe used 2 hESC lines  at passages 25-35to differentiate into a hepatic lineage according to theprotocol of Basma et al.  with somqRT-PCR results were converted to relative concentrations based on the standard curve method. Analysis of variances was performed on the readings from 60 samples and 48 different transcripts. We used the Statistical Analysis System (SAS) for 2-factor ANOVA by considering the hESC lines and the culture methods as 2 variable factors. P<0.01 were considered to be significant. SAS also provides a Duncan grouping chart for each gene in which samples are sorted based on that gene’s expression level. We used Eisen Lab and TreeView softwares for hierarchical clustering of the samples and genes.Clustering of the complete data set, as shown in Figure 1C, indicates that different hESC lines have similarexpression profiles at the undifferentiated stage. Once the cells start to spontaneously differentiate, their gene expression profiles differ significantly due to their distinct intrinsic lineage propensities. As expected, most of the genes that were markers of the same cell type were clustered in close proximity in the clustering tree, which indicated reliability of the results. Pluripotency markers Oct4, Nanog, and TDGF were all clustered together, as were the endoderm markers HNF3b, CXCR4, and SOX17.CHD1, a chromatin remodeler known to be involvedin formation and maintenance of the open chromatinstate, showed interesting results. We assessed the expression levels of CHD1 in 4 hESC samples, and at days 10 and 30 after spontaneous differentiation. Figure 2A shows that the expression level of CHD1 increased upon differentiation and peaked at day 10, which was unexpected if it had a similar role in mouse ESCs and hESCs. A study on mouse ESCs showed that this protein highly expressed in stem cells and was responsible for the existence of a completely open chromatin in undifferentiated stem cells, and was required for the stemness property of mESCs.A comparison of lineage-specificity among the 4 hESC lines in the current study was the first step for additional approval of the reliability of marker expression levels in spontaneous differentiation. The majority of the 46 examined markers showed significant differences in gene expression patterns among the 4 lines, which suggested divergent lineage specification. Based on spontaneous differentiation results, we selected 2 hESC lines for directed differentiation tests. The RH2 line showed the highest propensity to express endodermal  and hepPrimary results confirmed the effectiveness ofspontaneous differentiation in a comparison of the differenthESC lines. In the second step, we compared EB versusthe adherent culture methods using the same approach.Gene expression levels in suspension and adherent culturewere compared for all lines. Interestingly, different lineage markers showed differential patterns in each condition. The ectodermal, neural, endodermal, hepatic, and endothelial markers had higher expression levels in suspension condition , while tWe confirmed the spontaneous differentiation results by direct differentiation of the RH5 cell line into neural cells according to the 2 induction protocols. The first protocol comprised the suspension culture stage, whereas the second protocol used the adherent culture during differentiation. Results of directed differentiation of stem cells to neurons confirmed spontaneous differentiation results, which showed that the suspension protocol was significantly more effective than the adherent culture.Our results confirmed that the expression level of neural specific expressing markers like GFAP, PAX6, PDGFRa  and b-Tublin  are higher in suspension (EB form) than adherent system in both direct and spontaneous differentiation systems, even in neuro-ectodermal specification or neural maturation stages. Although in both differentiation systems we have acceptable increase in neural lineage specific markers  . Both parameters had a significant influence on the final results. Although the effect of dimension has been extensively discussed, thereare few reports that have compared different lineages in 2D versus 3D culture systems. More recently, the 3D culture systems have been developed to recapitulate human complicated organs (like nervous system) development and differentiation in in vitro system starting from human pluripotent stem cells (hPSCs) by organoid technology . Although many studies have been conducted to reveal the mechanisms of 3D differentiation in higher organs studies but there are still many questions to be addressed . In the We observed that the ectodermal, neural, endodermal, hepatic, and endothelial markers had higher expression levels in cells grown in the suspension condition, while skin-specific markers were expressed more in the adherent system. A possible explanation could be the similarity of the ex vivo environment to the natural extracellular environment for each cell or tissue type . KeratiGene expression profile analysis of hESCs duringdifferentiation is a simple and reliable approach to predicttheir lineage propensities. This cost effective method could provide very useful data over a short period of time instead of cultivation of different hESC lines, differentiating them to all possible lineages, and comparing them. In the current study, we have used this invaluable tool to compare the expression levels of lineage-specific markers during spontaneous differentiation of hESC lines in suspension versus adherent culture systems. Although reports have shown this tool’s usefulness, we decided to confirm spontaneous differentiation results by direct differentiation of the RH5 cell line to neural cells. The direct differentiation data verified previous results andproved that this tool could be useful for optimization of hESC differentiation. According to the current results,the addition of a suspension culture step in differentiation of most lineages would be necessary. This new strategymay lead to the optimization of some common protocols used for neural differentiation of stem cells, and providea standard platform for analysis of other types of differentiation protocols.Future studies, such as high-throughput analysis of the expression profiles on ESC lines during differentiation in adherent versus suspension culture conditions, are required for additional information in this area. Elucidation of the mechanisms that cause the early events of lineage specification under the 2 culture conditions is necessary. Commonly used differentiation protocols can be compared with respect to different environmental variables such as chemical and mechanical properties of the culture system to enhance the efficiency of stem cell differentiation toward a desired cell type, and further pave the way for stem cells to be used in a clinical setting.


SHANK variants in human patients. A similar diversity of neuropsychiatric-like phenotypes is described for numerous Shank2 and Shank3 knockout (KO) mouse lines. In this review, we will focus on and discuss the experimental results obtained from different, but genetically related and therefore comparable, Shank2 mouse models. First, we will describe the distinct SHANK2 variant-mediated neurodevelopmental and neuropsychiatric disorders in human patients. Then we will discuss the current knowledge of the expressed SHANK2 isoforms in the mouse, and we will describe the genetic strategies used for generating three conventional and seven conditional Shank2 mouse lines. The distinct impairments i.e., autistic-like and mania-like behavior and the alterations on the molecular, electrophysiological and behavioral levels will be compared between the different Shank2 mouse models. We will present our view as to why in these mouse models a spectrum of phenotypes can arise from similar Shank2 gene manipulations and how Shank2 mutant mice can be used and should be analyzed on the behavioral level in future research.The SHANK scaffolding proteins are important organizers for signaling proteins in the postsynapse of excitatory neurons. The functional significance of SHANK proteins becomes apparent by the wide spectrum of neurodevelopmental and neuropsychiatric disorders associated with  SHANK3 variant in human patients with ASD . In humans, three different genes, SHANK1, 2 and 3 produce several SHANK isoforms via internal promoters and alternative splice products  in the postsynaptic density. Cortactin, a molecule involved in actin polymerization, was also shown to bind to PRR  are significantly associated with autism spectrum disorders (ASD). After the first publication of a SHANK variant-associated neurodevelopmental and neuropsychiatric disorders. Distinct SHANK mutations can yield different protein products, with a specific combination of protein domains, different from that of the WT protein. This, in turn, can affect the potential protein-protein interactions, thus altering the role of the protein in neurons. In ASD patients, chromosomal deletions, translocations, gene duplications, gene fragment deletions and coding mutations in all three SHANK genes were identified (see https://gene.sfari.org) but the simple haploinsufficiency caused by the mutation is unlikely to explain the broad spectrum of phenotypes. In order to dissect the causal link between SHANK variants and the underlying molecular mechanisms of neurodevelopmental and neuropsychiatric disorders, numerous mouse models for “shankopathies” were generated (Table Shank3 knockout (KO) mouse lines revealed differences in social and repetitive behavior as well as in cognitive functions, similar to the distinct neuropsychiatric phenotypes of different patients. These behavioral inconsistencies were explained partially by the residual SHANK3 isoforms that could be expressed from the targeted Shank3 gene in mice , a correlation that cannot be stated as strong for SHANK3 variants  variant found in seven schizophrenic male brothers, for example, was inherited from a healthy mother .The mouse SHANK2 expression is strongest in the brain, but SHANK2 mRNA can also be PCR-amplified in most peripheral tissues with the exception of skeletal muscles and heart  and their dendrites, whereas Shank3 mRNA was found only in the cerebellar granular cells; and (ii) Shank2 mRNA expression in the hippocampus is restricted to the cell layer of principal neurons, while the signals for the Shank1 and Shank3 mRNA can be detected in the molecular layers as well  were generated. Both mouse models resembled two PDZ domain-microdeletions found in two human patients , exon 16 was deleted , the exons 15-16 encoding gene segment was replaced by a loxP site flanked inverse-oriented neomycin resistance (neo) selection marker , significant differences on the molecular, electrophysiological, synaptic composition and anatomical levels of Shank216Δex or Shank215-16Δex were evident  and synaptic properties such as the AMPA/NMDA ratio and long-term potentiation  and the other in GABAergic inhibitory neurons . Thus, in Shank2 KO mice, a residual expression of truncated proteins or mRNAs, which differs between the Shank2 mouse lines .In 2017, Lim et al.  for Cre expression. Thus, when exon 24 was deleted efficiently in 3 weeks old Δex24-CaMKI2a-CreShank2 mice, they didn’t show any hyperactivity, which was used as signature for the mania-like behavior of the global Δex24Shank2 mice , which enhances the NMDAR function via mGluR5 activation, rescued the NMDAR signaling impairments, as measured in hippocampal CA1 cells. The CDPPB-mediated recovery of NMDAR function in Δex15-16Shank2 mice was correlated with normalized social interaction of CDPPB treated Δex15-16Shank2 mice. However, hyperactivity and anxiety-like behaviors were not recovered  synapses , thus leading to a diversity of phenotypes. However, epigenetic factors as well as inconsistent analysis of the various Shank2 mouse lines cannot yet be completely ruled out.The different alterations in synaptic responses, synaptic plasticity, social interaction and exploration behavior, as well as therapeutic responses in in vivo analysis of certain SHANK2 isoforms in mice, certain exons or SHANK internal promoters could be eliminated or certain SHANK isoforms could be overexpressed by viruses or transgenes. New genetic engineering techniques can generate relatively quickly novel Shank2 mouse lines. However, in future studies with novel constitutive and conditional Shank2 mouse lines, the expression of the residual truncated isoforms should be analyzed in more details, and the full battery of behavioral analysis covering ASD, SCZ, mania and Attention Deficit Hyperactivity Disorder (ADHD) has to be applied, since the existing Shank2 mouse models showed very distinct, unpredictable phenotype despite very similar genetic manipulations. In addition, males and females and real littermates need to be compared in detail to unravel epigenetic, environmental and gender effects on the expression of the phenotypes. More importantly, the current Shank2 mouse models provided first insights into molecular and physiological changes underlying ASD-related symptoms. The Shank2 mutant mice showed that ASD-related symptoms can be diminished by pharmacological treatment of the mutant mice, which can guide future therapeutic strategies for ASD patients. Now the application of spatio-temporal removal of SHANK2 using cell-type specific Cre or CreERT2 mice can identify those neuronal connections and circuits that have the strongest impact on the expression of the ASD-like phenotype. Moreover, conditional Shank2 KO mice can help to dissect the neurodevelopmental vs. the transient deficiencies mediated by the SHANK depletion. Once the most crucial neuronal circuits and cell types are identified, those neuronal populations can be targeted and analyzed in great detail in behaving mice by novel physiological technologies, e.g., multicellular recordings and optogenetics, which allow the recording, visualization and manipulation of neuronal activity ensembles during social interaction, repetitive behavior, vocalization and cognition, or even during different phases of sleep. Due to the very distinct phenotypes of different Shank2 mouse lines, it will be exciting to see how their altered neuronal network activity is correlated with the impaired social behavior.Understanding the detailed function of the multitude of SHANK isoforms is one of the biggest challenges in cognitive neuroscience. The disturbed balance of different SHANK proteins within individual postsynapses significantly affects the plasticity of synapses, possibly by changing the composition of the postsynaptic scaffold, which translates to a rearrangement of the glutamate receptors and measurable alterations in the glutamatergic signaling. For All authors significantly contributed to the manuscript. AE and RS wrote the first draft and provided the figures, tables and supplementary information. GR critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stellate cells, either hepatic (HSCs) or pancreatic (PSCs), are a type of interstitial cells characterized by their ability to store retinoids in lipid vesicles. In pathological conditions both HSCs and PSCs lose their retinoid content and transform into fibroblast-like cells, contributing to the fibrogenic response. HSCs also participate in other functions including vasoregulation, drug detoxification, immunotolerance, and maintenance of the hepatocyte population. PSCs maintain pancreatic tissue architecture and regulate pancreatic exocrine function. Recently, PSCs have attracted the attention of researchers due to their interactions with pancreatic ductal adenocarcinoma cells. PSCs promote tumour growth and angiogenesis, and their fibrotic activity increases the resistance of pancreatic cancer to chemotherapy and radiation. We are reviewing the current literature concerning the role played by retinoids in the physiology and pathophysiology of the stellate cells, paying attention to their developmental aspects as well as the function of stellate cells in tissue repair and organ regeneration. Sternzellen”. They were overlooked for almost a century, until their rediscovery in 1971 by Kenjiro Wake [Stellate cells, also called Ito cells, were first described by Kupffer in 1876 as “iro Wake . These riro Wake . Pancreairo Wake ,4. Despiiro Wake , proteomIn pathological conditions, stellate cells from both liver and pancreas lose their lipidic droplets and transform into fibroblast-like cells, a phenotype able to synthesize large amounts of extracellular matrix, contributing to the fibrogenic response to tissue damage  Figure .HSCs account for 5–8% of all the hepatic cells . SecretiPSCs also display a number of relevant functions . For exaWe aim to review, in this article, the relevant information about the role played by retinoids in the physiology and pathophysiology of the stellate cells from liver and pancreas, paying attention to developmental aspects as well as the function of stellate cells in tissue repair and organ regeneration.The embryonic origin of HSCs was first related with the endoderm from a supposed common hepatoblast/HSC progenitor ,15,16. AHSCs start to accumulate retinoids during foetal life, as early as E13 (stage 13 days post coitum) in rat embryos ,25. ThesCre) can be crossed with gene-targeted mice where a stop sequence flanked by lox sites precedes a YPF cassette in the Rosa26 locus (Rosa26REYFP reporter). The offspring of these crosses, carrying both genetic modifications, will show permanent expression of YFP in Wt1-expressing cells and their lineage [The embryonic origin of PSCs is also related, at least in part, with the coelomic epithelium of the pancreas. Between E10.5 and E15.5 in mouse embryos, this epithelium expresses WT1 A, allowi lineage   is a marker of HSCs, and the number of cRBP-1 + HSCs was used in the liver human foetuses with congenital diaphragmatic hernia to search for a correlation with the growth of liver and lungs . The res−/− foetal liver than in controls at E10, but there are no differences at E12, suggesting that RXRα mediated signalling is dispensable from E12 on [HSCs can play some role also in the foetal liver phase of definitive haematopoiesis. HSCs are topologically associated to hematopoietic sites (the space of Disse) . Insulinm E12 on  (reviewem E12 on ).Like HSCs, PSCs possess the mechanisms to accumulate and metabolize retinoids, bioactive molecules that are involved in both, exocrine and endocrine pancreas development . Differeβ1 integrin and collagen I matrix interactions are required for maintaining foetal pancreatic stellate cell function and proliferation. In fact, culture of human foetal PSCs on collagen I stimulates their proliferation, and blocking of the β1 integrin reduces adhesion, migration and proliferation .Development of stellate cells has been studied in other animal models. In zebrafish, HSCs labelled with a hand2:EGFP (enhanced green fluorescent protein) reporter colonize the liver migrating from the lateral mesoderm after the sinusoidal endothelial cells . MigratiFibroblastoid progenitors of HSCs appear in the Disse’s space of 6-day-old chick embryos. Retinoid accumulation revealed by fluorescence of lipid droplets starts later, by 9 days of incubation. By the end of incubation, about a half of the perisinusoidal cells show retinoid-containing droplets .As stated in the introduction both HSCs and PSCs can be activated by extrinsic signals and transdifferentiate into a myofibroblast-like phenotype, with enhanced migratory and extracellular matrix secreting abilities and expression of smooth muscle cell α-actin  Figure . In chroRetinoids have a strong ability to modulate the activation of stellate cells in both liver and pancreas. However, the role played by RA signalling in the process of stellate cell activation is still poorly known ,46. For In normal pancreas, retinoic acid signalling is restricted to the islets and a few exocrine cells, as shown by the retinoic acid responsive element (RARE-LacZ) murine model. After induction of pancreatitis with caerulein, the acinar cells become responsive to RA. However, when pancreatic cancer is induced, RA signalling activity is not detected in the tumours or in their precursor lesions . This obAll these observations about the behaviour of PSCs are important because activated PSCs promote growth of pancreatic ductal adenocarcinoma (PDAC) and the excess of extracellular matrix  increases the resistance of pancreatic cancer to chemotherapy and radiation. PSCs indirectly stimulate tumour growth and metastasis by promoting angiogenesis. PSCs from the primary tumour have been localized even associated to metastatic foci where they facilitate the proliferation of tumour cells . RetThe Kras(G12D)/Trp53(R172H)/Pdx-1-Cre (KPC) mouse model of PDAC ,64 has bOpen issues that deserve attention are the changes in retinoid storage and metabolism during stellate cell activation, as well as the roles of HSCs during fibrosis resolution. About the first issue, it has been shown that the stored retinols are metabolized into RA, promoting a number of reparative RA-dependent pathways in hepatocytes and also in immune cells .Besides their fibrogenic activity, activated stellate cells secrete cytokines and growth factors that promote the regeneration of both liver and pancreas. RA released by activated HSCs can have a direct mitogenic effect on hepatocytes through the RXRα receptor, contributing to tissue regeneration  .A surprising role of HSCs in hepatic regeneration was suggested by Yang et al. . These aPSCs also play an essential role in the regeneration of the pancreas after partial pancreatectomy. Proliferation of acinar and islet cells increases when co-cultured with activated PSCs. Interestingly, this effect is dependent of the production of extracellular matrix by the PSC. In fact, inhibition of the collagen synthesis using a specific siRNA encapsulated in a vitamin A-coupled liposome blocked this mitogenic effect .As described above for HSCs, PSCs might also display a wide regenerative potential. Treatment of isolated PSCs with cytokines can induce the expression of hepatocyte markers. Transplantation of GFP-labelled, activated PSCs into partially hepatectomised rats showed differentiation into hepatocytes and cholangiocytes . HoweverThe importance of RA signalling in pancreatic regeneration has been emphasized by our recent study on the conditional deletion of WT1 in adult mice . DownregIn recent years both HSCs and PSCs have acquired great importance with respect to knowledge of the normal functions of the liver and pancreas, and also because of their roles in pathologies of these organs. The ability of these cells to store and metabolize retinoids seems to be highly related to their physiological and pathophysiological functions. The unique properties of these cells in the liver and pancreas homeostasis and repair raise the issue of their potential utility in cell therapy. A preclinical study has demonstrated that cotransplantation of HSCs with hepatocytes improves hepatocyte engraftment . We have


Introduction: As the prevalence of kidney disease continues to rise worldwide, there is accumulating evidence that kidney injury and dysfunction, whether acute or chronic, is associated with major adverse outcomes, including mortality. Meanwhile, effective therapeutic options in the treatment of acute kidney injury (AKI) and chronic kidney disease (CKD) have been sparse. Many of the effective treatments that are routinely utilized for different pathologies in patients without kidney disease have failed to demonstrate efficacy in those with renal dysfunction. Hence, there is an urgent need for discovery of novel pathways that can be targeted for innovative and effective clinical therapies in renal disease states.Discussion: There is now accumulating evidence that the endocannabinoid (EC) system plays a prominent role in normal renal homeostasis and function. In addition, numerous recent studies have described mechanisms through which alteration in the EC system can contribute to kidney damage and disease. These include a potential role for cannabinoid receptors in tubulo-glomerular damage and fibrosis, which are common features of AKI, interstitial nephritis, glomerulopathy, and other conditions leading to AKI and CKD.Conclusion: These findings suggest that manipulating the EC system may be an effective therapeutic strategy for the treatment of kidney disease and injury. However, further mechanistic studies are needed to fully delineate the role of this system in various conditions affecting the kidneys. Furthermore, while most of the current literature is focused on the role of the EC system as a whole in renal pathophysiology, future studies will also need to clarify the contribution of each component of this system, including the EC mediators, in the pathogenesis of kidney disease and their potential role as part of a therapeutic strategy. The kidneys play a central role in normal body homeostasis through a variety of functions, including removal of byproducts of metabolism, clearance of toxins, regulation of body volume status, electrolytes and systemic hemodynamics, and production of hormones such as erythropoietin and active vitamin D. Hence, it is not surprising that kidney damage is associated with significant morbidity and mortality. The latter is true whether the decline in renal function is part of an acute process such as acute kidney injury (AKI) due to tubular necrosis, or a more chronic process such as chronic kidney disease (CKD) caused by hypertension (HTN) or diabetes. Furthermore, the mechanisms responsible for renal injury are complex and can be varied. While these mechanisms are regularly categorized based on the type of injury (acute or chronic) and anatomic part of the nephron affected , there is significant overlap between these categories. For instance, there is evidence indicating that AKI can result in CKD. In addition, there is frequent overlap between the different anatomic sites of injury given that damage to one part of the nephron over a period of time can result in injury to other sites. For example, while diabetic kidney disease often manifests with glomerular injury and proteinuria, over an extended period of time it also results in tubulointerstitial damage and fibrosis leading to progressive CKD and end-stage kidney disease. Therefore, understanding the underlying pathways whose alterations can result in various forms of renal damage and injury can play an important role in devising effective therapies to prevent and treat kidney disease. In this regard, there is accumulating evidence that indicates that the endocannabinoid (EC) system plays a major role in normal renal physiology. In addition, there are data demonstrating that alterations of this pathway can lead to the pathogenesis of both acute and chronic kidney disease. Therefore, evaluation of the EC system can be a promising area of discovery, which may result in the generation of potentially novel therapies aimed at treating various forms of kidney disease.1 The most well-characterized ECs are N-arachidonoyl ethanolamide, also known as anandamide (AEA), and 2-arachidonoyl-sn-glycerol (2-AG).2 These lipid-derived molecules are generated on-demand by the metabolism of membrane phospholipids in response to various stimuli, including elevated intracellular calcium or metabotropic receptor activation.3 After production, they bind to the local cannabinoid receptors in an autocrine or paracrine manner, although measurable concentrations of these ligands can also be found in the blood, cerebrospinal fluid, and lymph.4 While the potential endocrine actions of these ECs remain an area of active research, it is well established that they act locally by binding with two widely studied cannabinoid receptors, cannabinoid subtype-1 (CB1) and subtype-2 (CB2).5 AEA and 2-AG can subsequently be taken up by cells through a high-affinity uptake mechanism7 and rapidly degraded through the action of the enzymes, fatty acid amide hydrolase (FAAH), and monoacylglycerol lipase (MGL), respectively.1The EC system comprises endogenous fatty acid-derived ligands, their receptors, and the enzymes required for their biosynthesis and degradation.9 The effects produced by the actions of this system in the normal and pathological conditions of the kidney, however, have not been fully delineated given the many complexities involved in the production and breakdown of EC ligands.8–11 In addition, the differential distribution and actions of the CB1 and CB2 receptors in various structures and cell subtypes in the kidney can ultimately result in varied signaling outcomes whose overall impact will be difficult to predict. Accordingly, identifying the physiologic and pathophysiologic roles of the EC system in the field of nephrology remains an active area of exploration.While the role of the EC system has been initially a focus of extensive research in the central nervous system, over the course of the past two decades, a significant number of studies have confirmed its presence and importance in the peripheral organs, including the kidneys. In this regard, substantial concentrations of ECs, the machinery required for their biosynthesis and degradation, as well as CB receptors have been detected in kidney tissue.1 and CB2 belong to a class of seven transmembrane domain G-protein-coupled receptors that are functionally dependent on the activation of heterotrimeric Gi/G0 proteins.11 Although the activation of both receptors results in the inhibition of adenylyl cyclase enzyme and increased activity of mitogen-activated protein kinase (MAPK), CB1 activation has also been shown to stimulate nitric oxide synthase and directly control the activation of ion channels. The latter include the inwardly rectifying and A-type outward potassium channels, D-type outward potassium channels, and N-type and P/Q-type calcium channels.13 Despite the common G-protein subunit shared between CB1 and CB2 receptors, their activation can produce opposing biological effects in normal and diseased states, in part due to the abundance and localization of these cannabinoid receptors and their EC ligands.It has been shown that CB1 receptor was initially thought to be localized to the central and peripheral nervous system,14 it has been shown to be present in peripheral organs such as the kidneys.16 For instance, the presence of functional CB1 receptor has been demonstrated in proximal convoluted tubules, distal tubules, and intercalated cells of the collecting duct in the human kidney13  of the loop of Henle,18 and glomeruli,19–23 as well as in various kidney cell subtypes such as glomerular podocytes,25 tubular epithelial cells,26–29 and cultured mesangial cells.31 Similarly, the expression of CB2 receptors, although previously thought to be predominantly in immune cells,32 has also been demonstrated in renal tissue.33 For example, CB2 receptor expression has been localized to podocytes,25 proximal tubule cells,34 and mesangial cells31 in human and rat renal cortex samples.While the CBkidney13 . Further35–39 While the renal cortex displayed similar levels of AEA and 2-AG, AEA was demonstrated to be enriched in the kidney medulla compared with the cortex, while the levels of 2-AG in the medulla were similar to those of both ECs in the cortex.39 Moreover, AEA is present in cultured renal endothelial and mesangial cells at low levels and can be synthesized from arachidonic acid and ethanolamine and catabolized by AEA amidase in these kidney cell subtypes.31 The expression of FAAH was shown to be augmented in the renal cortex  in comparison to its low expression levels in the medulla.39In addition to differential expression of CB receptors in different tissues and cells, the complex regulation of the biosynthesis and degradation of the kidney's high basal levels of ECs through downstream enzymes contributes to the varied signaling effects of these ligands.1 receptor.39–41 In the following sections, we describe some effects of EC system activation on renal physiologic function . The actions of the AEA signaling system are likely conducted through endothelial and mesangial cells, which are capable of producing and metabolizing AEA,31 as well as through the hyperpolarization of smooth muscle cells via the activation of potassium channels.42 It should be noted that there are also non-CB1 receptor–dependent mechanisms by which ECs can mediate a vasodilatory effect and thereby regulate renal hemodynamics.43 Future studies need to further elucidate the role of the latter mechanisms in normal renal physiologic homeostasis.Under normal physiologic conditions, the EC system plays a critical role in the regulation of renal hemodynamics. For instance, it was shown that intravenous administration of AEA decreased glomerular filtration rate and increased renal blood flow in rodents, independent of changes in blood pressure.1 receptor) was shown to stimulate nitric oxide production, leading to an inhibition of sodium transport through the apical Na+/H+ transporter and Na+/K+/2Cl− co-transporter. This was also associated with reduced oxygen consumption in the TAL portion of the nephron.18 This suggests that the activation of CB receptors via AEA can regulate renal blood flow as well as tubular handling of solutes, which can ultimately impact renal salt and water clearance.AEA has been shown to have a regulatory effect on tubular sodium transport. In the medullary TAL of Henle's loop, AEA  expression levels and subsequently reduce nephrin gene and protein levels, suggesting a potential pathway for podocyte dysregulation and proteinuria.40To examine the role of glomerular CBThe role of the EC system in renal pathology and dysfunction is an emerging area of research, which has been studied primarily in the context of CB receptors. Alterations of CB receptor expression and activity have been discovered in various renal diseases such as diabetic nephropathy, CKD, and different types of kidney injury . Collect45 Several studies have examined the role of the EC system in diabetes-related podocyte, mesangial and tubular cell injury, as well as the function of CB receptor activation on the adverse outcomes of diabetic nephropathy -induced mouse model of diabetic nephropathy, albuminuria was reduced as a result of CB1 receptor inverse agonist restored GFR, reduced proteinuria, and improved the markers of podocyte health through modulation of the renin–angiotensin system and inhibition of apoptosis.24In Zucker diabetic fatty (ZDF) rats, which develop type 2 diabetes due to obesity caused by a dysfunctional leptin receptor, chronic administration of a CB1 receptor in various parts of the nephron, there is also evidence that CB2 receptor expression is significantly reduced. For example, STZ-induced diabetic nephropathy in mice is associated with the downregulation of glomerular podocyte CB2 receptor expression.48 Similarly, there is decreased expression of the CB2 receptor in proximal tubule cells following exposure to elevated concentrations of albumin and glucose.34 Furthermore, CB2 receptor activation has been shown to ameliorate albuminuria, restore podocyte protein expression, reduce monocyte infiltration, and decrease the expression of renal profibrotic markers33 in rats with obesity-related nephropathy. CB2 agonism in obese diabetic nephropathy BTBR ob/ob mouse strain also reduced albuminuria, ameliorated dysfunctional nephrin expression in podocytes, and reduced mesangial matrix expansion, fibronectin accumulation, and sclerotic damage.49While diabetic kidney disease is associated with increased expression of the CB1 receptors and activation of CB2 receptors using selective pharmacological ligands is associated with the restoration of renal structure and function, specifically albuminuria and the expression of inflammatory markers, in genetic and experimental models of diabetic nephropathy.These studies demonstrate that antagonism of CB50 with obese individuals possessing a higher risk of progressing to end-stage renal disease.51 A study by Jenkin et al.33 revealed a role of CB2 receptor activation in reducing the progression of obesity-related kidney dysfunction by decreasing proteinuria, creatinine clearance, and renal fibrotic markers. In contrast, ZDF rats treated with a CB1 inverse agonist showed improved renal structure and function.24 Surprisingly, these rats also displayed a marked increase in body weight compared with the weight stability of control mice, which was thought to be due to the development of extreme hyperglycemia in vehicle-treated controls.24 In a separate study, CB1 antagonism was shown to decrease albuminuria, reduce mesangial expansion, and ameliorate the expression of profibrotic and proinflammatory kidney proteins in lean and obese diabetic mouse models.23 These findings suggest that the consequences of CB1 modulation in diabetes can differ based on the experimental model, presence of obesity, and presence of hyperglycemia.Obesity is associated with and acts as a risk factor for the development of diabetic nephropathy,1 receptor expression in the kidney is notably upregulated, and treatment with a CB1 receptor antagonist reduced weight, systolic blood pressure, plasma leptin, albuminuria, and plasma creatinine levels. This is associated with the amelioration of glomerulopathy.16 Furthermore, studies using obese Zucker rats demonstrated that the CB1 inverse agonist, rimonabant, ameliorated proteinuria in an animal model of obesity-induced nephropathy.21 Treatment with rimonabant partially restored creatinine clearance, reduced glomerulosclerosis and tubular-interstitial fibrosis, and lowered tubular damage and renal hypertrophy.21 It should also be noted that these findings may have been mediated by the effects of rimonabant and not related to the EC system. While obesity in fa/fa Zucker rats is caused by a mutation of the leptin receptor, rimonabant acts to increase leptin uptake by the kidney, which has been shown to reduce proximal tubule metabolic activity.52 Therefore, improvement in renal function in these rats may have occurred due to mechanisms related to leptin's role in proximal tubule cell metabolism,52–54 as opposed to a direct action on the EC system.In other studies that utilized rats with diet-induced obesity, CB1 receptors in renal proximal tubule cells, Udi et al.55 found that CB1 receptor deletion did not protect the mice from the deleterious metabolic effects associated with obesity, but significantly diminished obesity-induced lipid accumulation in the kidney. Furthermore, the stimulation of CB1 receptors in renal proximal tubule cells was found to be associated with decreased activation of liver kinase B1 and decreased activity of AMP-activated protein kinase, as well as reduced fatty acid beta-oxidation.55 These findings indicate a potential relationship between renal proximal tubular epithelial cell CB1 receptor and the pathologic effects of obesity-induced renal lipotoxicity and nephropathy.Using a novel mouse strain lacking CB1 receptor highlight its partial potential in acting as a therapeutic target for obesity-induced renal disease. Further studies are needed to ascertain the efficacy of modulating CB1 in the kidney to improve renal dysfunction independent of its effects on weight.In summary, the findings related to the CB1 receptor has been shown to be upregulated in other renal disorders marked by interstitial inflammation and fibrosis, including acute interstitial nephritis.20 Using unilateral ureteral obstruction (UUO) as an experimental model for renal fibrosis in mice, Lecru et al.20 showed that CB1 receptor expression was upregulated in UUO animals compared with controls. This is also associated with a marked increase in the renal content of 2-AG. Treatment of UUO mice with rimonabant reduced monocyte chemoattractant protein-1 synthesis and decreased macrophage infiltration.20 It was also shown that CB1 receptor activation led to enhanced VEGF levels, which subsequently reduced nephrin expression and protein levels.40The CB1 and CB2 receptors and their modulation in the pathogenesis of various forms of AKI. With regard to ischemic AKI, selective CB1 and CB2 receptor agonists were found to have a dose-dependent effect in preventing tubular damage following renal ischemia/reperfusion injury in mouse kidney.56 In a separate study, however, the administration of cannabidiol, a non-psychoactive constituent of cannabis with poorly defined pharmacological properties, led to a reduction in renal tubular injury in rats following bilateral renal ischemia/reperfusion.57 Cannabidiol significantly attenuated the elevation of serum creatinine and renal malondialdehyde and nitric oxide levels associated with this condition.57 In a more recent study, a triazolopyrimidine-derived CB2 receptor agonist was demonstrated to play a protective role in inflammatory renal injury following bilateral kidney ischemia/reperfusion.58There is accumulating evidence indicating the important role of CB1 and the protective effects of CB2 activation on a nephrotoxic model of AKI in cisplatin-induced renal injury.59–61 Inhibiting CB1 receptor35 or activating CB2 receptor60 limited oxidative stress and inflammation and reduced tubular damage in kidneys of animals with cisplatin-induced AKI. In addition, ß-Caryophyllene, a natural agonist of CB2 receptor, dose-dependently protected against the deleterious effects of cisplatin-induced nephrotoxicity.61A series of studies have demonstrated the deleterious role of CB1 and CB2 receptors have been shown to play a role in renal apoptotic and inflammatory signaling pathways.60 Indeed, activating CB1 receptors is known to result in enhanced expression of oxidative/nitrosative stress markers, which activate p38, MAPK, and c-Jun N-terminal kinase pathways, as well as nuclear factor kappa-light-chain-enhancer of activated B cells-dependent transcription of downstream proinflammatory target genes. Ultimately, the activation of either route leads to apoptotic cell death and inflammation in the kidney.35 Conversely, CB2 receptor activation has been found to reduce proapoptotic signaling60 and mediate anti-inflammatory effects by attenuating immune cell infiltrates and inflammatory cytokine release.59 Mukhopadhyay et al.59 showed that a peripherally restricted CB2 receptor agonist (LEI-101), in a mouse model of cisplatin-induced nephrotoxicity, dose-dependently attenuated renal dysfunction as measured by serum concentrations of blood urea nitrogen (BUN) and creatinine. The protective effects of CB2 receptor activation in these studies were absent in CB2 receptor knockout mice, suggesting that CB2 receptors are a promising therapeutic target for reducing renal inflammation, oxidative/nitrosative stress, and apoptosis.Furthermore, CB63 In a study using a cecal ligation and puncture (CLP) mouse model of sepsis, CB2 receptor knockout mice demonstrated increased mortality, lung injury, bacteremia, neutrophil recruitment, and decreased p38 MAPK activity at the site of infection.64 Treatment with a selective CB2 receptor agonist reduced the effects caused by CLP, such as inflammation, lung damage, and neutrophil recruitment, and ultimately improved survival.64 These findings are in line with evidence demonstrating that following CB2 localization to leukocytes, their activation has been shown to mitigate leukocyte tumor necrosis factor-α-induced endothelial cell activation, adhesion and migration of leukocytes, as well as proinflammatory modulators.65–68 Therefore, CB2 receptor modulation may represent a novel therapeutic target in the treatment of SA-AKI.70Another major contributor to AKI, which is associated with significant morbidity and mortality, is sepsis-associated kidney injury (SA-AKI).71 as well as differences in the physiological outcome of receptor activation are likely related to the type of AKI and to the abundance and localization of receptors.The mechanism(s) by which the cannabinoid receptors modulate or recover tubular cell survival following acute damage are not well defined at this time. However, molecular differences in cannabinoid receptor mRNA and protein levels9; however, physiological responses elicited by these ligands under normal or pathological conditions have not been fully elucidated. Furthermore, detailed studies on how elevated or decreased levels of these ligands may impact renal function and pathology are scarce. For example, it is well known that AEA plays a role in the modulation of renal hemodynamics.31 Infusion of this ligand was found to be associated with vasorelaxation of juxtamedullary afferent arterioles in vitro,31 increased renal blood flow in rodents,17 and alteration of tubular sodium transport.18 While these effects may be partly mediated through the activation of CB1 and CB2 receptors, it is important to highlight that these findings indicate the total effect of this ligand and it is difficult to identify exactly which receptors are activated in each segment of the nephron. Furthermore, there are CB receptor–independent effects that are not accounted for when the role of these ligands were to be assessed only in the context of CB receptors.While many of the studies evaluating the role of the EC system in renal homeostasis and pathophysiology focused on CB receptors and their modulation, it is important to keep in mind that the overall effects of activation and inhibition of the EC system are dependent on various factors, only a portion of which is related to the activity of CB receptors. For example, the chief endogenous activators of the CB receptors, AEA and 2-AG, are present in substantial concentrations in the kidney72 described alterations to the EC system in primary and secondary HTN, noting that these conditions resulted in renal oxidative stress through increased reactive oxygen species (ROS) and diminished levels of antioxidant enzymes. Despite the enhanced activity of FAAH and MGL in primary and secondary hypertensive rats, the levels of AEA and 2-AG in the kidney were significantly increased.72 Increasing endogenous levels of AEA by pharmacologically inhibiting its degradative enzyme, FAAH, with a selective FAAH inhibitor, URB597, was found to have resulted in the inhibition of ROS generation in both types of hypertensive rats. These effects were mediated through improvement in antioxidant defense in the primary spontaneously hypertensive rat (SHR) kidney via the Nrf2 pathway, as well as through reduced proinflammatory responses in secondary hypertensive  rats.72 Furthermore, URB597 augmented ROS-dependent phospholipid peroxidation products and levels of ECs in both types of hypertensive kidneys, which resulted in enhanced CB receptor expression in SHR rats and enhanced expression of CB2 and TRPV1 receptors in DOCA-salt rats.72 Chronic treatment of Wistar normotensive control rats with URB597 similarly enhanced phospholipid oxidation in the kidney, comparable to its administration in DOCA-salt rats.72 Thus, while the EC system appears to play a protective role in HTN, the administration of a FAAH inhibitor did not significantly alter the proinflammatory or oxidative conditions caused by primary HTN, and only created imbalances between ECs, oxidants, and proinflammatory factors in secondary HTN, potentially leading to the development of kidney dysfunction.Recent studies have begun to address this important point by attempting to define the impact of these ligands in renal disease states. Biernacki et al.73 demonstrated that renal ischemia/reperfusion injury is associated with a significant increase in renal 2-AG content using a bilateral ischemia/reperfusion mouse model of AKI. It was found that the augmentation of kidney 2-AG concentrations following MGL inhibitor administration resulted in improved serum BUN, creatinine, and tubular damage score; however, the mRNA gene expression of renal inflammation and oxidative stress markers was not altered. Conversely, in a cisplatin-induced nephrotoxic model of AKI, cisplatin enhanced AEA but not 2-AG levels in renal tissue.35With regard to other renal conditions, such as AKI, studies have shown varied responses to kidney injury in EC expression levels. Moradi et al.1 receptor activation in mediating glomerular podocyte injury. Jourdan et al.74 showed that chronic exposure of human cultured podocytes to high glucose resulted in a significant upregulation in CB1 receptor gene expression, which is also associated with an increase in cellular AEA and 2-AG. This is associated with signs of inflammation and podocyte injury, which manifest as decreased podocin and nephrin and increased desmin gene expression.74 In contrast, Li et al.75 reported the protective functions of AEA following l-homocysteine (Hcys)-induced podocyte injury. AEA blocked Hcys-induced NLRP3 inflammasome activation in cultured podocytes and ameliorated podocyte dysfunction, ultimately precluding glomerular damage.75 Therefore, while the former study demonstrated that an increase in CB1 receptor gene expression accompanied by an upregulation in AEA and 2-AG is associated with podocyte injury, the latter study suggests that AEA exerts protective and anti-inflammatory effects in podocytes. Future studies are needed to investigate the role of EC ligands in CB receptor activation under varied conditions in renal health and disease.To date, the mechanisms and conditions under which CB receptors are activated by ECs in the kidney—and subsequently the signaling cascades that result from this activation—have not been fully described. Studies have demonstrated conflicting results describing the role of AEA and CB1 and CB2 receptors and their major physiologic activators (AEA and 2-AG), have been localized to a variety of renal cell subtypes across different species. Consequently, the activation or inhibition of CB1 and CB2 can significantly impact renal function with beneficial or adverse effects. Altered CB receptor expression has been demonstrated in a number of renal diseases, including nephropathy, CKD, and AKI. These findings have led to the investigation of CB receptor manipulation using pharmacological agents, which have partly pointed to the CB receptors as potential therapeutic targets for renal dysfunction. An important result of these studies was the demonstration that CB1 and CB2 receptors act via separate pathways and modulate distinct downstream targets in the kidney, despite a largely homogenous distribution in the renal system.The EC system has been found to regulate a variety of functions in renal health and disease states. Various components of the EC system, namely the CBMore recently, the EC system has been studied for its association with a variety of renal disease states. Collectively, these studies suggest that the activity of ECs should be examined separately from their interactions with CB receptors, as conflicting results were seen in the biological responses elicited by ECs and the activation of their receptors.76 Thus, while current data suggest that modulating EC system function and activity may provide a viable therapeutic intervention for renal dysfunction, future studies are essential to further elucidate the mechanisms through which ECs and CB receptors participate in renal physiology and disease, as well as the clinical context in which their stimulation or suppression could lead to beneficial or deleterious effects in the kidney.In summary, significant focus has been placed on evaluating the role of CB receptors in renal function, homeostasis, and pathophysiology. While these endeavors have contributed significantly to our understanding of the role of the EC system in the kidney, important areas of opportunity remain for future research, especially the role of EC ligands as mediators of EC system activity. At present, their role in renal physiology and pathophysiology remains to be fully elucidated. Furthermore, the clinical implications and relevance of EC system alteration will need to be further evaluated.


Inflammation is a common factor of pathologies such as obesity, type 2 diabetes or neurodegenerative diseases. Chronic inflammation is considered part of the pathogenic mechanisms of different disorders associated with aging. Interestingly, peripheral inflammation and the associated metabolic alterations not only facilitate insulin resistance and diabetes but also neurodegenerative disorders. Therefore, the identification of novel pathways, common to the development of these diseases, which modulate the immune response and signaling is key. It will provide highly relevant information to advance our knowledge of the multifactorial process of aging, and to establish new biomarkers and/or therapeutic targets to counteract the underlying chronic inflammatory processes. One novel pathway that regulates peripheral and central immune responses is triggered by the cytokines pleiotrophin (PTN) and midkine (MK), which bind its receptor, Receptor Protein Tyrosine Phosphatase (RPTP) β/ζ, and inactivate its phosphatase activity. In this review, we compile a growing body of knowledge suggesting that PTN and MK modulate the immune response and/or inflammation in different pathologies characterized by peripheral inflammation associated with insulin resistance, such as aging, and in central disorders characterized by overt neuroinflammation, such as neurodegenerative diseases and endotoxemia. Evidence strongly suggests that regulation of the PTN and MK signaling pathways may provide new therapeutic opportunities particularly in those neurological disorders characterized by increased PTN and/or MK cerebral levels and neuroinflammation. Importantly, we discuss existing therapeutics, and others being developed, that modulate these signaling pathways, and their potential use in pathologies characterized by overt neuroinflammation. Activation of the innate immunity in the Central Nervous System (CNS) is a critical step in the healing process after brain injury. However, cumulative evidence points to deleterious effects of chronic neuroinflammation . ActivatPtn/Mk developmental gene family  and induction of expression of inflammation-related molecules such as cytokines, have been described in detail. Although the discovery of new modulators of neuroinflammation can potentially translate in novel targets and biomarkers for different diseases, the genetic bases of neuroinflammation are still far from being fully understood. Recently, we identified Pleiotrophin (PTN) and Midkine (MK) as two novel neurotrophic factors that modulate neuroinflammation in different contexts . Pleiotre family . The pate family . They are family . As sugge family . Accordie family . Midkinee family . In addie family . It has  pathway , which c pathway . Other als of PD . Taken tMk-/- mice. In contrast, lipopolysaccharide (LPS)-induced striatal astrogliosis was blocked by genetic inactivation of Mk, suggesting a differential regulation of astrocytosis by MK depending on the inflammatory stimulus (Ptn overexpression in the brain (Ptn-Tg mice) potentiates the striatal astrocytosis induced by acute administrations of amphetamine  have been related with vascular injury and angiogenesis  and PTN ischemia . The angischemia . On the ischemia . As in tischemia . Thus, bMidkine and PTN bind different receptors such as Receptor Protein Tyrosine Phosphatase (RPTP) β/ζ , syndecaRPTPβ/ζ  , is abunThese signaling pathways triggered by PTN and MK through RPTPβ/ζ support important roles of these cytokines in neuroinflammation  since thPtn in developing and adult tissues, including nervous system and peripheral organs, has been described in detail  after in vitro and in vivo validation  than in subcutaneous WAT , including exosomes, are released by all cells, including those of adipose tissue and nervous system. Their capacity to deliver a variety of bioactive molecules  to both nearby and distal cells suggests that exosomes and specific miRNAs play a role in cancer, diabetes, obesity, cardiovascular disease  and CNS Since adipocytes secrete large proportions of exosomes, it has been proposed that exosomes and miRNAs derived from fat play a role in pathologies related with insulin resistance . FurtherPleiotrophin and MK are novel modulators of neuroinflammation that play different roles depending on the inflammatory stimulus and the duration of the neuroinflammatory processes. Further studies are needed to fully understand the role of these cytokines on neuroinflammation, particularly in chronic neuroinflammation. However, in the light of evidence summarized here, it is reasonable to hypothesize that both potentiation and inhibition of the PTN/MK signaling pathways are novel therapeutic strategies to modulate neuroinflammation in acute and chronic pathological states. For instance, potentiation of the acute neuroinflammatory actions of these cytokines may exert therapeutic effects in different types of brain injury through limitation of the damage and improvement of brain repair. In terms of metainflammation, potentiation of PTN/MK signaling pathways may contribute to control the inflammatory condition associated to metabolic disorders. On the contrary, inhibiting the actions of these cytokines may be beneficial in chronic neuroinflammatory states that promotes exacerbation of neurodegeneration.The administration of rMK and rPTN has been proposed in a number of CNS and peripheral injuries, rending already significant effects in preclinical models, particularly in the case of myocardial ischemia  and boneThe potential of therapeutic inhibition of MK and PTN has been studied in depth for the treatment of malignant diseases in different experimental models, including siRNA, shRNA, antibodies and RNA aptamers . InteresAlthough promising, the above mentioned strategies to potentiate or inhibit the actions of PTN and MK in neuroinflammation in CNS disorders are commonly restricted by the limitation of the route of administration . Thus, these effective therapeutic strategies in preclinical models of different diseases are not likely to progress through clinical development in the midterm. We will now summarize the existing modulators of PTN/MK signaling with approved therapeutic uses in humans and other promising strategies at the preclinical level that are more likely to progress through clinical development in the midterm.in vitro and in vivo. Interestingly, systemic administration of MY10 reduces alcohol consumption and blocks the rewarding effects of alcohol in mice (Ptn-Tg) overexpressing in the brain Ptn, the endogenous inhibitor of RPTPβ/ζ .Fyn kinase is a substrate of RPTPβ/ζ . Fyn hasSmall-molecule midkine inhibitors have been investigated mainly in the field of cancer therapy . A promiThe expression of the components of the PTN-MK-RPTPβ/ζ axis in immune cells and in inflammatory diseases suggests important roles for this axis in inflammation. Pleiotrophin has been recently identified as a limiting factor of metainflammation, a chronic pathological state that contributes to neuroinflammation and neurodegeneration. Pleiotrophin also seems to potentiate acute neuroinflammation independently of the inflammatory stimulus while MK seems to play different -even opposite- roles in acute neuroinflammation depending on the stimulus. Which are the functions of MK and PTN in chronic neuroinflammation is still a question of great biologic interest.For its pattern of expression and its known signaling cascades involving important regulators of inflammation as Fyn kinase and ALK, RPTPβ/ζ is a target receptor for PTN and MK in neuroinflammation. Pharmacologic modulation of the PTN, MK, RPTPβ/ζ and/or its downstream effectors, Fyn and ALK, is a novel therapeutic strategy to modulate neuroinflammation, from central or peripheral origin, in different pathological contexts.GH and EG wrote the manuscript and built The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Several studies focused on the identification of DNA mutations generated, or selected, during the derivation of hiPSCs, some of which are known to drive cancer formation. Avoiding such stable genomic aberrations is paramount for successful use of hiPSCs, but it is equally important to ensure that their epigenetic information is correct, given the critical role of epigenetics in transcriptional regulation and its involvement in a plethora of pathologic conditions. In this review we will focus on genomic imprinting, a prototypical epigenetic mechanism whereby a gene is expressed in a parent-of-origin specific manner, thanks to the differential methylation of specific DNA sequences. Conventional hiPSCs are thought to be in a pluripotent state primed for differentiation. They display a hypermethylated genome with an unexpected loss of DNA methylation at imprinted loci. Several groups recently reported the generation of hiPSCs in a more primitive developmental stage, called naïve pluripotency. Naïve hiPSCs share several features with early human embryos, such as a global genome hypomethylation, which is also accompanied by a widespread loss of DNA methylation at imprinted loci. Given that loss of imprinting has been observed in genetic developmental disorders as well as in a wide range of cancers, it is fundamental to make sure that hiPSCs do not show such epigenetic aberrations. We will discuss what specific imprinted genes, associated with human pathologies, have been found commonly misregulated in hiPSCs and suggest strategies to effectively detect and avoid such undesirable epigenetic abnormalities.Human induced Pluripotent Stem Cells (hiPSCs) have the capacity to generate a wide range of somatic cells, thus representing an ideal tool for regenerative medicine. Patient-derived hiPSCs are also used for  Human induced pluripotent stem cells (hiPSCs) display the important properties of long-term self-renewal and pluripotency: they are theoretically capable of generating unlimited amounts of any differentiated cell of the human body . For theThere are three possible sources of genetic alterations in iPSCs :(a)During the reprogramming process some pre-existing abnormalities, such as somatic mutations may be selected and expanded;(b)per se;The reprogramming process might generate rare alterations (c)In vitro expansion of hiPSCs for extended passaging might cause the generation of advantageous mutations.An extensive study conducted on hiPSCs generated from different types of donor cells found a similar mutation rate for both coding and non-coding regions, arguing against a functional role for such mutations . Ruiz anSardo and colleagues measured the rate of mutations in blood cells and hiPSCs derived from them. Despite a correlation between donor age and the number of mutations observed, there was no evidence for positive selection of somatic mutations in hiPSC, with a large degree of heterogeneity in the somatic mutations identified between lines derived from the same individual . A similIn sum, genetic mutations observed in hiPSCs are in part pre-existing abnormalities of source somatic cells that are passively fixed by the process of reprogramming.Induced pluripotent stem cells were originally generated using retrovirus-mediated delivery of reprogramming factors , but stade novo-generated CNVs, human retroviral or piggyBac generated hiPSCs lines were compared to human ESCs lines and parental fibroblasts lines  in culture. Not only cells spontaneously acquired P53 mutations, but the fraction of cells carrying the mutant P53 allele increased with passage number under standard culture conditions , causing the insufficient silencing of lineage-specific genes or the negative regulation of pluripotency genes , limitinOlder cells are more resistant to complete reprogramming , probablThe analysis of the DNA methylation profile of cells reprogrammed with different methods revealed profound differences. Somatic cell nuclear transfer (SCNT) is able to generate reprogrammed cells displaying a DNA methylation profile comparable to embryo-derived ESCs, while hiPSCs reprogrammed via the Yamanaka factors show a divergent profile, with memory of the somatic cell methylome  Ma et a. A possiThe reprogramming process frequently generates aberrantly methylated regions that are dissimilar to the somatic donor cell and the human ESCs : these rWhile extended culturing exposes cells to the risk of accumulation of genetic defects, prolonged expansion seems to allow the loss of the epigenetic memory, thus reducing the differences between hiPSCs and hESCs . At the XIST .The majority of the autosomal genes are expressed from both copies; however, a small subset of genes has one copy turned off in a parent-of-origin dependent manner. These genes are called ‘imprinted.’ In this class of genes one allele is marked with DNA methylation. Imprinted genes acquire DNA methylation during gametogenesis and maintain their DNA methylation profile stable in all adult tissues . Thus, tImprinted genes usually form clusters, where several coding and at least one non-coding RNA are under the control of one unique DMR. These DMRs can be intergenic or intragenic. Interestingly, paternal DMRs are few in number and all intergenic, while maternal DMRs are more abundant and all reside within genes . GeneticThe loss or gain of DNA methylation over DMRs can both result in the monoallelic to biallelic conversion of gene expression or in the repression of the only transcriptionally active allele. This leads to alterations in the dosage of the imprinted transcripts that are potentially harmful. Indeed, the monoallelic expression of imprinted genes allows a tight control of their dosage and is essential for the proper development of the embryo .H19, IGF2, MEG3, PEG3, PEG10, MEST  are more susceptible to LOI than hESCs : this suin vitro, have been shown to induce a significant increase in the occurrence of imprinting disorders like Beckwith–Wiedemann syndrome in babies conceived using such techniques  found within the imprinted DMRs (reviewed in diabetes . If the diabetes . It woulThe loss of imprinting observed in naïve hiPSCs could be the combined effect of:(1)Expansion of pre-existing LOI: in (2)per se. In order to evaluate such hypothesis, low passage naïve hiPSCs should be compared to matching fibroblasts, to see if the DNA methylation profile of imprinted genes has undergone any change during the reprogramming process and consequently if their monoallelic expression has been maintained or lost.The reprogramming process itself: as observed in primed hiPSCs, the process of reprogramming could generate imprinting abnormalities (3)A result of the extensive passaging, cell expansion and selection: over several rounds of cell divisions, a cell with global low levels of DNA methylation could easily undergo LOI. To investigate this, low- and high-passage naïve cells should be compared to see if there is any difference in the levels of DNA methylation and if some imprinted genes acquired biallelic expression.Although additional analyses will be needed to tease out the contribution of the three possible mechanisms listed above, we should also discuss studies performed in mouse naïve pluripotent stem cells. When mouse naïve pluripotent stem cells are expanded in serum-free medium containing two inhibitors of the MEK and GSK3 kinases and the cytokine LIF (2i+LIF), they show a hypomethylated genome, as also reported for pluripotent cells of the pre-implantation embryo . In contEven though global DNA methylation levels substantially decrease over a relatively short time when cells are passed from Serum+LIF to 2i+LIF cultures, imprints are rather stable in the same time frame and upon extended passaging . This hoIn sum, imprinting at methylated loci is more labile in female murine cells than male cells, but we do not know if the same holds true in human cells. Moreover, extensive culture leads to LOI and optimization of the media composition could improve imprinting stability also in human cells.Imprinted genes are dosage-sensitive: their deregulated expression leads to different pathologies, ranging from cancer to developmental diseases known also as imprinting disorders .In this section we summarize the pathologies linked to imprinted genes, focusing on those genes more often found misregulated in hiPSCS. Before hiPSCs are used for cell replacement, we should be aware of how LOI could compromise the function of specific cell types. Imprinted genes are organized in clusters under the control of single DMRs, therefore changes in DNA methylation of a DMR will result in altered expression of multiple imprinted genes . The majIn humans, six imprinted clusters have been consistently associated with disease:(1) IC1 (H19/IGF2)(2) IC2 (KvDMR)(3) DLK1/MEG3(4) SNURF/SNRPN(5) GNAS(6) PLAGL1/HYMAI.H19, IGF2, KCNQ10T1, MEG3 , Russel-Silver syndrome  and Uniparental disomy 14 (MEG3) and is also observed in several tumors see .Despite the fact that BWS and RSS can be caused by mutations or LOI occurring in genes that belong to the same cluster, BWS and RSS show opposite phenotypes, with BWS characterized by fetal and extraembryonic overgrowth, while RSS is characterized by severe pre- and post-natal growth retardation .KCNQ10T1/CDKN1C cluster , RTL1as (RTL1 antisense), and MEG8 (see DLK1-GTL2 intergenic DMR (IG-DMR) and the GTL2-DMR are extensively hypermethylated in the paternal allele and grossly hypomethylated in the maternal one. Deregulation of the genes within the DLK1-MEG3 imprinted cluster on chromosome 14q32 is responsible for the distinct phenotypes observed in the patients of maternal and paternal UPD14 syndromes Individuals affected by imprinting disorders are usually more prone to develop cancer.(2)Loss of imprinting either by genetic mutations or epimutations, is a very common phenomenon seen in cancer, and it often is an early event .Cell replacement therapies, whereby somatic cells of interest are generated from pluripotent stem cells, are currently under clinical trials see , for conGuhr et al., 2018MEG3 LOI is linked to skeletal abnormalities [see section Uniparental Disomy 14 (UPD14)], thus hiPSCs displaying altered MEG3 expression should not be used for the derivation of mesenchymal stem cells, that are progenitors of osteocytes. Similarly, because of the implication of LOI in cancer, hiPSCs with alteration of the DLK1 DMR should not be used for the derivation of dopaminergic neurons or oligodendrocytes (DLK1 LOI is linked to neuroblastomas and gliomas) or cells showing LOI at the IC1 DMR (H19-IGF2) should not be used for generating pancreatic cells (IGF2 LOI is associated to pancreatic cancer).We believe that hiPSCs showing imprinting defects should not be used for cell replacement therapies. For instance, A screening on the hiPSCs population, first for misregulated expression of imprinted transcripts, possibly followed by analyses of genetic mutations and epimutations, should be performed when choosing hiPSCs for cell replacement therapies. Additional safety measures could be considered, as in the case of the most advanced clinincal trials based on hESC-derived pancreatic beta cells, whereby cells are encapsulated before transplantation, in order to protect the host from potential harmful features of the cells.Loss of imprinting has been often measured in terms of loss of DNA methylation, but this does not always result in a loss of monoallelic expression, because other mechanisms  besides DNA methylation can regulate the expression of the silenced allele . FurtherGiven that appropriate differentiation and proliferation of hiPSCs could be affected by the dose of imprinted gene transcripts, an effective way to measure LOI would be the measurement of levels of expression and the distinction of the mono- versus bi-allelic expression .In particular, the two following analyses should be performed:(1)Measuring the expression levels of imprinted genes, relative to a panel of control cells where imprinting is stable. This type of analysis can be easily applied to novel hiPSCs generated either by quantitative RT-PCR or by RNA-sequencing.(2)Detection of monoallelic vs. biallelic expression based on SNPs . For thiin vitro expansion and the reprogramming process contribute to LOI. Moreover, hiPSCs in a naïve state of pluripotency display more profound alterations at the level of methylation of imprinting control regions, while additional analyses will be needed to gauge the impact on the expression of imprinted genes.Loss of imprinting is observed in somatic cells in culture and, more frequently, in iPSCs, indicating that both the Additional studies will be also needed to optimize culture conditions and reprogramming protocols in order to generate hiPSCs with more stable imprinting. At the same time such studies might shed light on the mechanisms of imprinting maintenance. For example, maternal and paternal imprints appear specifically more stable in primed and naïve hiPSCs  respectiIn conclusion, hiPSCs represent an ideal tool for the study of regulation of genomic imprinting and, at the same time, a better understanding of the mechanisms controlling imprinted gene expression will have an impact on safety of hiPSC applications.No datasets were generated or analyzed for this study.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.