The current leaders in the commercialization of organoids, their geographical location, and application areas.
\r\n\tThis book intends to provide the reader with a comprehensive overview of the current state-of-the-art novel imaging techniques by focusing on the most important evidence-based developments in this area.
",isbn:null,printIsbn:null,pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,isNomenclature:!1,hash:"d9159ce31733bf78cc2a79b18c225994",bookSignature:"Dr. Gabriel Cismaru",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11867.jpg",keywords:"Hypertrophic Cardiomyopathy, Dilated Cardiomyopathy, Restrictive Cardiomyopathy, Transesophageal Echocardiography, Intracardiac Echocardiography, 3-Dimensional Echocardiography, Adult Congenital Heart Disease, Tetralogy of Fallot, Transposition of the Great Vessels, Coronary Artery Disease, Risk Stratification, Revascularization",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"April 21st 2022",dateEndSecondStepPublish:"May 19th 2022",dateEndThirdStepPublish:"July 18th 2022",dateEndFourthStepPublish:"October 6th 2022",dateEndFifthStepPublish:"December 5th 2022",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"3 months",secondStepPassed:!0,areRegistrationsClosed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Dr. Cismaru Gabriel is an Assistant Professor at the University of Medicine and Pharmacy Cluj-Napoca, certified in Cardiology. After completing his certification in cardiology, Dr. Cismaru began his electrophysiology fellowship at the Institut Lorrain du Coeur et des Vaisseaux Louis Mathieu. He has authored or co-authored peer-reviewed articles and book chapters in the field of cardiac pacing, defibrillation, electrophysiological study, and catheter ablation.",coeditorOneBiosketch:"Raluca Tomoaia is an MD, Ph.D. in novel techniques in Echocardiography at the University of Medicine and Pharmacy in Cluj-Napoca, Romania., assistant professor, and a researcher in echocardiography and cardiovascular imaging.",coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"191888",title:"Dr.",name:"Gabriel",middleName:null,surname:"Cismaru",slug:"gabriel-cismaru",fullName:"Gabriel Cismaru",profilePictureURL:"https://mts.intechopen.com/storage/users/191888/images/system/191888.png",biography:"Dr. Cismaru Gabriel is an assistant professor at the Cluj-Napoca University of Medicine and Pharmacy, Romania, where he has been qualified in cardiology since 2011. He obtained his Ph.D. in medicine with a research thesis on electrophysiology and pro-arrhythmic drugs in 2016. Dr. Cismaru began his electrophysiology fellowship at the Institut Lorrain du Coeur et des Vaisseaux Louis Mathieu, France, after finishing his cardiology certification with stages in Clermont-Ferrand and Dinan, France. He began working at the Rehabilitation Hospital\\'s Electrophysiology Laboratory in Cluj-Napoca in 2011. He is an experienced operator who can implant pacemakers, CRTs, and ICDs, as well as perform catheter ablation of supraventricular and ventricular arrhythmias such as ventricular tachycardia and ventricular fibrillation. He has been qualified in pediatric cardiology since 2022, and he regularly performs device implantation and catheter ablation in children. Dr. Cismaru has authored or co-authored peer-reviewed publications and book chapters on cardiac pacing, defibrillation, electrophysiological studies, and catheter ablation.",institutionString:"Iuliu Hațieganu University of Medicine and Pharmacy",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"7",totalChapterViews:"0",totalEditedBooks:"2",institution:{name:"Iuliu Hațieganu University of Medicine and Pharmacy",institutionURL:null,country:{name:"Romania"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:null},relatedBooks:[{type:"book",id:"5970",title:"Bedside Procedures",subtitle:null,isOpenForSubmission:!1,hash:"ba56d3036ac823a7155f40e4a02c030d",slug:"bedside-procedures",bookSignature:"Gabriel Cismaru",coverURL:"https://cdn.intechopen.com/books/images_new/5970.jpg",editedByType:"Edited by",editors:[{id:"191888",title:"Dr.",name:"Gabriel",surname:"Cismaru",slug:"gabriel-cismaru",fullName:"Gabriel Cismaru"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"9064",title:"Epidemiology and Treatment of Atrial Fibrillation",subtitle:null,isOpenForSubmission:!1,hash:"1cd6bf2b3181eb82446347fbe478a2bc",slug:"epidemiology-and-treatment-of-atrial-fibrillation",bookSignature:"Gabriel Cismaru and Keith Andrew Chan",coverURL:"https://cdn.intechopen.com/books/images_new/9064.jpg",editedByType:"Edited by",editors:[{id:"191888",title:"Dr.",name:"Gabriel",surname:"Cismaru",slug:"gabriel-cismaru",fullName:"Gabriel Cismaru"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"2270",title:"Fourier Transform",subtitle:"Materials Analysis",isOpenForSubmission:!1,hash:"5e094b066da527193e878e160b4772af",slug:"fourier-transform-materials-analysis",bookSignature:"Salih Mohammed Salih",coverURL:"https://cdn.intechopen.com/books/images_new/2270.jpg",editedByType:"Edited by",editors:[{id:"111691",title:"Dr.Ing.",name:"Salih",surname:"Salih",slug:"salih-salih",fullName:"Salih Salih"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"42965",title:"MicroRNAs Regulated Brain Tumor Cell Phenotype and Their Therapeutic Potential",doi:"10.5772/52362",slug:"micrornas-regulated-brain-tumor-cell-phenotype-and-their-therapeutic-potential",body:'MicroRNAs (miRNAs)are short 18–25 nucleotide small non-coding RNA molecules that function to silence gene expression via sophisticated post-transcriptional regulation[1]. Since their discovery in the early 1990s, these small molecules have been shown to play an important regulatory role in a wide range of biological and pathological processes. Over 30% of human messenger RNAs (mRNAs) are regulated by miRNAs[2]. miRNAs generated by the canonical biogenesis pathway are transcribed as precursor RNAs from intergenic, intronic or polycistronic genomic loci by RNA polymerase II (Pol II). The primary miRNA (pri-miRNA) transcript forms a stem–loop structure that is recognized and processed by the Drosha and DGCR8 RNase III complex or the spliceosome apparatus in the nucleus. In the non-canonical miRNA pathway, miRNAs are transcribed directly as endogenous short hairpin RNAs (endo-shRNAs) or derive directly through splicing from introns that can refold into hairpins (mirtrons). The trimmed precursor (pre-miRNA) hairpins from both canonical and non-canonical miRNA pathways are then transported by an exportin 5 and RAN-GTP-dependent process to the cytosol, where they are typically further processed by the Dicer and transactivation-response RNA-binding protein (TRBP) RNase III enzyme complex to form the mature double-stranded ~22-nucleotide miRNA. Argonaute proteins (for example, AGO2)
then unwind the miRNA duplex and facilitate incorporation of the miRNA-targeting strand (also known as the guide strand) into the AGO-containing RNA-induced silencing complex (RISC). The RISC– miRNA assembly is then guided to specific target sequences in mRNAs. The initial recognition of mRNAs by the RISC–miRNA complex is driven primarily by Watson–Crick base-pairing of nucleotides 2 to 8 in the mature miRNA (termed the seed sequence) with specific mRNA target sequences chiefly located in the 3′ untranslated region, and additional base-pairing affords greater affinity and targeting efficiency(Figure1)[3].
Given the pivotal regulatory role of miRNAs in a broad range of biological processes, it is not surprising that miRNAs play a role in human cancers, including brain tumor. First, about 50% of human miRNAs are located in cancer-associated genomic regions and fragile sites, suggesting that they might be the target genes underlying such aberrant intervals [5]. Second, the advent of high-throughput detection method has promoted expression profiling of miRNAs in normal and tumor tissues. Compared to normal tissues, anomalous levels of miRNA subsets have been found in almost all tumor types examined [6, 7]. Third, miRNAs with tumor-suppressive gene and oncogene-like properties have been described.
Since the first description of aberrant miRNA expression in glioblastomas and pituitary adenomas in 2005 [8, 9], there have been increasing reports each year about miRNA deregulation and function in various brain tumors. In this chapter, we summarize the current findings of miRNA study in brain cancers and discuss the diagnostic and therapeutic potential of miRNAs, mainly in glioma.
The miRNA biogenesis pathway[
CBTRUS obtained incidence data from 48 population-based cancer registries that include cases of malignant and non–malignant (benign and uncertain) primary brain and central nervous system tumors in the United States in 2004-2007. The most frequently reported histology is the predominately non–malignant meningioma, which accounts for 34% of all tumors, followed by glioblastoma (17%). The predominately non-malignant pituitary and nerve sheath tumors account for 13% and 9% of all tumors, respectively. Acoustic neuromas (defined by ICD-O-3 site code C72.4 and histology code 9560) account for 63% of all nerve sheath tumors(Figure2).
Only recently, the miRNAs attracted increasing attention as potential diagnostic or even therapeutic tools in brain tumors. Profiling techniques to identify global expression patterns of miRNAs in brain tumors have been widely used to uncover aberrantly expressed microRNAs in tumor genomes. Ciafrè et al. found nine (miR-10b, miR-130a, miR-221, miR-125b-1, miR-125b-2, miR-9-2, miR-21, miR-25, miR-123) and four miRNAs (miR-128a, miR-181c, miR-181a, miR-181b), respectively, out of 245 miRNAs to be up-/down-regulated in human glioblastoma samples, and nine (miR-221, miR-23a, miR-24-2, miR-24-1, miR-23b, miR-21,miR-222-prec, miR-191, miR-220) and seven miRNAs(miR-181a, miR-181b, miR-128b, miR-197, miR-181c,miR-125b-2, miR-125b-1), respectively, to be up-/down-regulated in human glioblastoma cell lines[10]. Chan et al.[8] demonstrated five (miR-21, miR-138, miR-347, miR-291-5′, miR-135) and three miRNAs (miR-198, miR-188,miR-202), respectively, out of 180 miRNAs to be up- and downregulated in glioblastoma samples. Sasayama et al.[11] found miR-10b, miR-21, miR-183, miR-92b and miR-106b to be up-, and miR- 302c*, miR-379, miR-329, miR-134 and miR-369-3p to be downregulated in human glioblastoma samples. Other studies reported several miRNAs to be significantly deregulated in glioma samples of Chinese patients (including miR-34a, miR-15b, miR-200a and miR-146b) [12], or miR-29b, miR-125a and miR-149 to be downregulated in glioblastomas [13]. In an array study with 192 miRNAs, 13 miRNAs (miR-101, miR-128a, miR-132, miR-133a, miR-133b, miR-149, miR-153, miR-154*, miR-185, miR-29b, miR-323, miR-328, miR-330) were found to be downregulated and three miRNAs to be upregulated (miR-21, miR-155, miR-210) in glioblastoma multiforme [14]. Another microarray study identified 55 miRNAs out of 756 miRNAs to be upregulated and 29miRNAs to be downregulated in malignant astrocytomas (primary and secondary glioblastoma and anaplastic astrocytoma, respectively) compared to controls [15].
Distribution of All Primary Brain and CNS Tumors by Histology (N=226,791) CBTRUS Statistical Report: NPCR and SEER Data from 2004-2007
In additional to gliomas, miRNA profiling also had been discovered in some other brain tumors. Ferretti et al were the first to identify signatures of a set of 248 miRNAs in a panel of primary medulloblastomas and normal cerebellar controls using high throughput expression profiles. They showed different expression profiles between normal brain and tumor and between distinct tumor histotypes. In particular, they detected an upregulation of mir-21 and miR-17-92 cluster (miR-17-5p, miR-20a and miR-19a) and a downregulation of miR-128a/b, let-7, miR-124a, miR-103, miR-134, miR-138, miR-149, miR-181b, miR-9 and miR-125a, most of them previously reported to be dysregulated in other brain tumor cell lines or nervous system cancers [16]. Moreover, Recent microarray data reported a possible role of miRNAs in pituitary adenomas. The first connection between pituitary adenomas and miRNAs was established by Bottoni et al in 2005, that showed a downregulation of mir-15a and miR-16-1 in GH-secreting and in PRL-secreting adenomas compared to normal pituitary tissue[8]. In 2007 Bottoni et al explored the miRNAome of pituitary tumors by microarray. They found that 30 miRNAs are differentially expressed between normal pituitary gland and pituitary adenomas. Among them, miR-150, miR-152, miR-191, and miR-192 were found to be upregulated in pituitary adenomas, and miR-132, miR-128a, miR-136, miR-16-1, and let-7 are downregulated[17]. As for meningioma, Feng Zhi et al found a list of 14 miRNAs that were differentially expressed in meningioma compared to normal adjacent tissue(NAT)samples. Twelve miRNAs, including miR-17-5p, miR-22-3p, miR-24-3p, miR-26b-5p, miR-27a-3p, miR-27b-3p, miR-96-5p, miR-146a-5p, miR-155-5p, miR-186-5p, miR-190a and miR-199a were shown to be upregulated by a factor greater than twofold, whereas two miRNAs, including miR-29c-3p and miR-219-5p, were significantly downregulated[18].
The use of miRNAs as tumor biomarkers has gained growing interest in the last few years. Accumulating evidence indicates that miRNA expression can be used as a prognostic and/or diagnostic marker for brain tumor patients. Niyazi et al. separated 35 glioblastoma patients into long- and short-term survivors. Then, they found that some miRNAs were significantly different in two group, including miR-3163, miR-539, miR-1305, miR-1260 and let-7a. this is the first dataset defining a prognostic role of miRNA expression patterns in patients with glioblastoma[19]. Moreover, Ma also identified that MiR-196b is overexpressed and confers a poor prognosis via promoting cellular proliferation in glioblastoma patients[20]. Costa et al identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and downregulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival[21].
Increasing evidence supports the supposition that miRNAs play an important role in different types of cancers and in various aspects of cancer biology. Abnormal miR levels in tumors have important pathogenetic consequences: miRNAs may act as oncogenes or suppressor genes[22]. For examples, Calin et al. [23] identified two clustered miRNAs (miR-15a and miR-16-1) on the minimal deletion region of chromosome 13q14 and showed that the levels of these miRNAs were significantly reduced in the majority (68%) of B-cell chronic lymphocytic leukemia (CLL). Both miRNAs target the anti-apoptotic gene BCL2, which is frequently overexpressed in CLL [24]. These findings indicate that downregulation of miR-15a and miR-16-1 elevates BCL2 level, contributing to CLL formation and suggesting a tumor-suppressive role for both miRNAs. In contrast, miR-17-92 showed marked upregulation in B-cell lymphomas. Enforced expression of miR-17-92 cluster acted with c-myc to accelerate the onset of B-cell lymphoma in a mouse model [25]. The data suggest that miR-17-92 cluster is a potential oncogene.
Recent evidence supports the ability of miRNAs to regulate brain tumor cell phenotype. We had done some work to identified that miR-221 and miR-222 regulated their target expression to co-modulated glioma cell phenotype, including proliferation, cell cycle, apoptosis and invasion(Figure3)[26-28], and so on.
miR-221 and miR-222 regulated their target expression to co-modulated glioma cell phenotype. (A: MTT assay show that knockdown of miR-221/222 lowed proliferated rate of glioma cell; B: knockdown of miR-221/222 induce G1 arrest in glioma cell ;C: Annexin V analysis showed that knockdown of miR-221/222 significantly increased glioma cell apoptosis; D: The transwell assay revealed that knockdown of miR-221/222 significantly decreased glioma cell invasion.
MiRNAs also regulated radio- and chemo-resistence of glioma. Hierarchical clustering analysis of expression 1100 miRNAs in three glioma cell lines treated with clinically relevant doses of radiation (2Gy) revealed significant (3-4 fold) up-regulation of several miRNA that are implicated in stimulation of survival and proliferation of tumor cells [29]. The set of up-regulated miRNAs includes miR-1285, miR-151-5p, and miR-24-1 that display beneficial effects on tumors by inhibiting the core tumor suppressor p53 (miR-1285) and supporting migration, local metastasis (miR-151-5p), and antiapoptosis (miR-24-1) [30]. Overall, activation of these miRNAs might possibly increase tumor radioresistance in subsequent radiotherapy sessions and stimulate motility of cancer cells thereby at least partially explaining the evidence on enhanced migration of malignant glioma cells in response to radiotherapy [31]. The radiation treatment of glioma cell lines with normal capacity to repair radiation-induced double strand breaks (DSB) of DNA caused activation of let-7 [29], a family of miRNA that suppresses proliferation of gliomblastoma cells [30] (Fig. 4). In contrast, in the radiosensitive human glioma cell line M059J that is deficient in DNA-dependent protein kinase (DNA-PK) and has a low activity of ATM, two key members of the non-homologous end joining pathway of DNA-DSB repair, let-7 miRNA was down-regulated [30].
Moreover, MiRNAs were shown to be also involved in the regulation of chemoresistence of glioma by modulating some relevant proteins that are involved in drug metabolism and drug transporter. MiRNAs also affect chemotherapeutic agents induced DNA damage repair (Fig. 4).
MicroRNAs are involved in the regulation of DNA repair in cancer cells after treatment with ionizing radiation (radiotherapy) and DNA alkylating drugs such as temozolomide and carmistine[
MiRNAs affect biological character of varies cells by regulating their targets. So, deregulated miRNAs regulated brain tumor cell phenotype by regulating their targets, including proliferation, cell cycle, apoptosis, invasion, and so on.
MiR-221 and miR-222 are both up-regulated in several glioma derived cell lines and in glioma samples [26-28]. They are clustered within a 1-kb genomic interval on chromosome X, so they could be generated from the same primary transcript [26].
Two members of the Kip/Cip family of CDK inhibitors and key negative regulators of the cell cycle, p57 and p27, have been identified as putative direct targets of these miRNAs. According to this theory, the 3\'-UTR of p57 harbors one site expected to be recognized by both miR-221 and miR-222, whereas the 3\'-UTR of p27 contains two sites for both miRNAs. Transfection of both miR-221 and miR-222 greatly reduce the levels of p57 and p27 proteins, while downregulation of either microRNA 221 or 222 in glioblastoma cells caused an increase in p27 levels and enhanced expression of the luciferase reporter gene fused to the p27 3\'-UTR. These results suggest that miR-221 and miR-222 could promote cell proliferation and S-phase entry together with further cooperative events and that miRNA 221 and 222 inhibition may be a potential therapeutic target to reduce the aggressive growth of glioblastoma by restoring normal levels of their target proteins [32]. The inverse correlation between mir-221/222 and p27 levels, the impairment of growth potential and the G1 to S shift in the cell cycle of glioma cells after inhibition of mir-221/222, were later confirmed by Zhang et al. They extended their study to in vivo trials and showed that miR-221/222 knocked-down through antisense oligonucleotides strongly reduced glioma subcutaneous mice xenografts growth through up regulation of p27 [26] and enhanced radiosensitivity of glioblastoma cell lines [33]. Further evidence showed that knockdown of miR-221/222 induced a change of mitochondrial membrane potential and caspase-mediated apoptosis on glioblastoma cells. Indeed, mir-221/222 directly downregulate the proapoptotic protein PUMA, which leads in turn to decrease Bcl-2 and increase BAX. these results confirm the oncogenic role of mir-221/222 on glioblastoma [27]. Our recent studies identified that mir-221/222 increased cell invasion and reduced gap junction intercellular communication (GJIC) by regulating TIMP3 and Cx43[28,34].
Microarray profiling studies, further validated by Northern blotting qRT-PCR and real-time PCR analysis, showed that miRNA-21 is strongly elevated in glioma and glioblastoma tumor samples and glioma cell lines when compared to non-neoplastic control samples, and upregulation is particularly prominent in grade IV astrocytomas [35-37]. High expression of miR-21 was significantly associated with poor patient survival, suggesting that, in combination with other aberrant expressed miRNAs (low expression of miR-181b or miR-106a), miRNA-21 profiling has potential application as novel diagnostic and prognostic indicator [37].
Suppression of mir-21 in glioblastoma cells decreased the metabolic activity of cell culture and cell number, and was associated with a marked increase in apoptosis and caspase activation [35]. MiR-21 knockdown also leads to a considerable reduction of glioma volumes in mouse xenografts [38]. Knockdown mir-21 inhibited proliferation and cell invasion and induced apoptosis through the activation of caspase-3 and -9 in glioma cell lines, which may be related to a mir-21-dependent modulation of multiple potential target genes, such as TIMP3 [39,40].
From an in silico constructed study of miR-21 predicted targets and further pathway analysis of computer generated lists of the identified target genes, emerged that mir-21 targets multiple components of the p53, transforming growth factor-β (TGF-β) and mitochondrial apoptosis pathways, that contribute to its tumor promoting and antiapoptotic activity. The phenotypic effects observed upon downregulation of miR-21 in glioblastoma cells, reflect the repression of these pathways and result in significant increase in apoptotic cells, reduced growth and cell cycle arrest at G0-G1 [41]. Further bioinformatics analysis evidenced that multiple genes involved in apoptosis pathways (such as PDCD4, MTAP, and SOX5), carry putative miR-21 binding sites. PDCD4 mRNA is a direct functional target of miR-21 and its expression inversely correlates with mir-21 in a number of glioblastoma cell lines (T98G, A172, U87, and U251). Consistent with these observations, downregulation of mir-21 restored protein level of PDCD4 while, ectopic overexpression of mir-21, inhibits PDCD4-dependent apoptosis [42]. Mir-21 controls tumor invasiveness and microvascular proliferation by regulating the expression of two of the major inhibitors of matrix metalloproteinases (MMPs): RECK and TIMP3. Mir-21 knockdown decreases RECK and TIMP3 protein levels and MMPs activity both in vivo and in vitro leading to a reduction of glioma cell motility and invasion [43]. Although PTEN tumor suppressor gene has been validated as a miR-21 target by computational analysis, down-regulation of mir-21 leads to an inhibition of tumor growth in glioblastoma cell lines and xenograft tumors independently of PTEN mutational status [44,45]. MiRNA expression profile scanning data after inhibition of miR-21, strongly indicated that BID, FAS, PRS6, and SOCS4 tumor suppressor genes were upregulated and evidenced a suppression of EGFR, activated Akt, cyclin D, and Bcl-2. This study highlighted that miR-21 targets multiple pathways responsible of inhibition of glioma growth in absence of PTEN [46].
Further findings strengthened the hypothesis that miR-21 represents a promising target to improve the efficacy of chemotherapy. In a recent study mir-21 was shown to play a key role in promoting human glioblastoma cells U87MG resistance against the antitumoral agent temozolomide (TMZ). Indeed, ectopic overexpression of mir-21 significantly reduced TMZ-induced apoptosis in this cell line through a suppression of Bax/Bcl-2 ratio and caspase-3 activity. These results confirm the hypothesis that mir-21 overexpression during glioma progression could be responsible of clinical resistance to chemotherapy with the promising alkylating agent TMZ [47]. Li et al demonstrated that mir-21 is also involved in glioblastoma cell chemoresistance to the chemotherapeutic agent VM-26, as shown by the observation that miR-21 knockdown sensitized GBM cells to VM-26. This is likely to happen because mir-21 regulates and inhibit LRRFIP1 gene expression by direct interaction [48]. This gene encodes a protein, also known as the TRAF-interacting protein (TRIP), that inhibits NF-κB signaling, a pathway that is known to be responsible for protection against apoptosis [49] and tumor chemoresistance [50]. Additionally, suppression of miRNA-21 expression in glioblastoma cell lines enhances sensitivity of cancer cells to antineoplastic cytotoxic therapy with neural precursor cells (NPC) expressing a secretable variant of the cytotoxic agent tumor necrosis factor related apoptosis inducing ligand (S-TRAIL). The synergistic effect was observed both in vitro, through an increased caspase associated cytotoxic death, and in vivo, where miR-21 knockdown and NPC-mediated S-TRAIL reduce cancer growth in tumor xenografts [38]. A recent study that evaluated the potential role of mir-21 as a therapeutic tool to enhance the cytotoxic effect of standard chemotherapy showed that co-delivery of miR-21 and 5-fluorouracil (5-FU) by using a poly(amidoamine) (PAMAM) dendrimer as a carrier, significantly improved the cytotoxicity of the antitumor agent leading to an higher apoptosis rate and a reduction of the migration ability of tumor cells [51]. Finally, downregulation of miR-21, contributes to the antitumor effects of IFN-β on glioma cell and intracranial tumor xenografts and the activation of the transcription factor STAT3 may have a key role in the IFN-β mediated suppression of mir-21 [52].
Mir-34a, transcriptional target of p53 located within chromosome 1p36, has been proposed as a potential tumor suppressor. MiR-34a is also downregulated in glioblastoma tissues and cell lines compared to normal brain tissues and is markedly reduced in p53-mutant cells compared to cells expressing wild-type p53 [53,54].
MiR-34a possesses hundreds of predicted mRNA targets which could mediate its inhibitory effects on tumor growth. A few of these have been experimentally verified and include oncogenes such as MYC, CCND1, CDK6, SIRT1 [55] and c-Met [56]. It was shown to directly inhibit c-Met in glioma and medulloblastoma cells and Notch-1/Notch-2 in glioma cells and stem cells by binding to the 3\'-UTRs of their mRNA. Furthermore, mir-34a inversely correlates with c-Met, Notch1, Notch2 and CDK6 protein expression in glioma cells. Transfection of microRNA34a in brain tumor cells strongly inhibits cell proliferation, cell cycle progression, cell survival, cell invasion, and in vivo glioma xenograft growth but do not affect cell cycle or death when transfected to human astrocytes, which showed normal expression levels of microRNA 34a. Restoration of c-Met or Notch-1 and Notch-2 expression by constructs lacking 3\'-UTR regions partially reverted cell cycle arrest and apoptosis induced by miR-34a in glioma cells or stem cells, confirming the hypothesis that the antitumor effects of miR-34a are achieved via targeting of multiple oncogenes [53]. Interestingly, mir-34a also affect glioma stem cell differentiation and growth. Luan et al recently reported silent information regulator 1 (Sirt1) as a direct negative target of mir34a in glioma cell lines at a posttranscriptional level [54]. Sirt1 is a NAD-dependent deacetylase that regulates apoptosis in response to oxidative and genotoxic stress, and has recently been involved in tumorigenesis acting as an oncogene [57].
Mir-128, a brain enriched microRNA, is downregulated in glioma cell lines and tissue when compared to normal brain samples [58]. Mir-128 overexpression decreased glioma cell proliferation in vitro and glioma xenograft growth in vivo, and is inversely correlated to Bmi-1 expression. Mir-128 directly targets and downregulates Bmi-1 by binding its 3\'-UTR region [59] and leads to a concomitant overexpression of p21CIP1 and a decrease in phosphorylated Akt [58]. Bmi-1 expression is a critical factor of normal stem cell maintenance and glioblastoma self-renewal and loss of Bmi-1-mediated self-renewal of neural stem cells has been shown to be associated with upregulation of p21CIP1 [60] and decreased Akt activation [61]. Consistently with these observations, miR-128 overexpression in human glioma neurosphere cultures possessing a glioma ‘stem-like’ cell phenotype, evidenced that miR-128 specifically blocked glioma self-renewal reducing neurosphere number and size. In conclusion, mir-128 downregulation is likely to enhance glioma tumorigenesis by promoting an undifferentiated phenotype and self renewing state through Bmi-1 increased expression [58]. By bioinformatical analysis, E2F3a, a transcription factor involved in cell cycle progression [62], has been identified as a direct target of this miRNA. Indeed, Mir-128 and E2F3a levels are negatively correlated and mir-128 overexpression has similar inhibitory effects on proliferation of glioma cell lines as E2F3a knocking down Ectopic overexpression of E2F3a partially reversed the effects of mir-128, suggesting that mir-128 could exert its antitumor effects at least partially by inhibiting E2F3a expression. Among its target genes angiopoietin-related protein 5 (ARP5) was identified with bioinformatical tools and then confirmed to be inversely correlated to mir-128 levels in glioblastoma cells and tissue and downregulated after ectopic overexpression of this miRNA [58]. ARP5 seems to be a key regulator of cell proliferation, remodeling and regeneration [63] and could be a member of a group of genes regulated by mir-128 that coordinately contribute to glioma and GBM pathology [59].
Mir-451 was found to be upregulated in glioma samples compared to normal brain on microarray-based miRNAome profiling [64]. MiR-451 showed a striking spatial distribution, with groups of positive cells concentrated around a subset of blood vessels. Moreover, high levels of mir-451 are correlated to a poor prognosis in glioma patients. Mir-451 could regulate the balance of proliferation and migration in glioma cells in response to changes in glucose levels and metabolic stress. It regulates 5\'-adenosine monophosphate activated protein kinase (AMPK) pathway in response to glucose levels in glioma cells, through the modulation of the activity of its upstream activator LKB1 [65]. Activation of LKB1 is a potent anti-proliferative signal, and also influences cell polarity, a process known to affect cell motility [66].
MiR-451 regulates LKB1 activity by directly targeting CAB39, a component of the active LKB1complex, leading to a downregulation of CAB39 protein levels. Down-regulation of mir-451 levels in response to glucose deprivation leads to intense activation of LKB1 and downstream pathways, allowing cells to survive metabolic stress and promoting cancer cell migration. Conversely, when glucose is sufficient, elevated miR-451 levels lead to reduced LKB1/AMPK pathway activation. This facilitates cell proliferation but makes cells more sensitive to glucose deprivation. Thus mir-451 could be a key regulator of adaptive response of cancer cells to altered energy availability [65].
In partial contrast with these studies a recent report found mir-451 to be downregulated in glioma cell lines. Transfection of mir-451 to glioma cell lines was able to inhibit cell growth, induced G0/G1 phase arrest, increased cell apoptosis and most notably diminished the invasive capacity of these cells as evidenced by transwell invasion assay. These tumor suppressive effects may be due to modulated expression of a panel of proteins including CyclinD1, p27, MMP 2/9 and Bcl-2, probably via regulation of the PI3K/AKT signaling pathway. Indeed, expression of Akt1 protein decreased after mir-451 transfection [67].
Mir-7, a miRNA modulated during neural differentiation of embryonic stem cells is a putative tumor suppressor gene in glioblastoma. It inhibits EGFR gene expression by directly binding its mRNA at 3\'-UTR. Furthermore, miRNA-7 suppresses the activation of Akt pathway and reduces phospho-Akt levels by directly targeting its upstream regulators IRS-1 and IRS-2 [68]. They also identified Raf1 as a direct target of miR-7 by microarray analysis, Western blot and luciferase reporter assays of glioblastoma cells transfected with miR-7 [69]. Inhibition of Akt activation seems to occur independently of mir-7-mediated repression of EGFR pathway. Consistenly with these findings miRNA-7 was markedly downregulated in glioblastoma tissue when compared to normal brain. The proposed mechanism that explains the lower expression of this miRNA is a processing defect in generating pre-miR-7 from pri-miR-7 in glioblastoma. Pre-miRNA-7 transfection decreased viability and invasiveness of glioblastoma lines and glioblastoma derived stem cells and led to an increase in the sub-G0 apoptotic fraction, a decrease in the S fraction and determined a G2-M arrest, most likely by affecting EGFR and Akt pathways, which have an established key role in gliomagenesis [68]. Indeed, EGFR is frequently amplified and highly expressed in glioblastomas [26]. In a recent report mir-7 was also shown to directly inhibit p21-activated kinase 1 (Pak1) in non-brain cancer cells [70]. Pak1 is a widely upregulated signaling kinase in multiple human cancers, and it is involved in the regulation of many signaling pathways, including EGFR and Akt, thus interfering with cell motility, proliferation, and apoptosis [71]. Furthermore, in glioblastomas, PAK1 upregulation is a negative prognostic marker and its knockdown results in impaired cell invasion [72]. These preliminary results suggest mir-7 could have a potential and promising therapeutic role in brain tumors.
Mir-181a and mir-181b are well known brain-enriched miRNAs [73]. Downregulation of mir-181a and mir-181b was detected in both glioma samples and cell lines [74]. Transfection of both these miRNAs on glioma cell lines results in cell growth inhibition, invasion reduction and apoptosis induction, miR-181b being more effective than miR-181a [74]. In a recent miRNA expression profiling conducted on a set of 124 astrocytoma samples and 60 normal adjacent tissue samples by qRT-PCR, Zhi et al found that downregulation of miR-181b is significantly associated with poor patient survival independently of other clinicopathological factors. Thus, mir-181b has a great potential for being used as a diagnostic and prognostic marker and to better select patients for adjuvant therapy [39].
In a recent study MiR-181b and miR-181c downregulation was found to be significantly associated to a positive response to concomitant chemoradiotherapy with temozolomide in glioblastoma patients, suggesting they could be used as predictive factors for therapy efficacy [75].
MiRNA-124 and miRNA-137 are downregulated in anaplastic astrocytomas and glioblastoma multiforme relative to non-neoplastic brain tissue [9-10]. Consequently, growth factor signaling could promote brain tumor formation through suppression of miR-124 and/or miR-137 expression and inhibition of neural stem cells/tumor stem cell differentiation. Transfection of mir-124 and mir-137 to neural stem cells and tumor-derived stem cells leads to induction of cellular markers of differentiation, G1 cell cycle arrest and reductions of the expression of the cell cycle regulator CDK6, a direct target of both miRNAs. These changes are accompanied by reduced self-renewal and tumorigenicity. Overexpression of miR-124 or miR-137 also reduced the expression of phosphorylated RB, a downstream target of CDK6 [76]. These results suggest that ectopic expression of miRNA-124 and miRNA-137 could represent a valid therapy for glioblastoma multiforme treatment inducing differentiation of tumor cells and cell cycle arrest.
Mir-125b downregulated in glioma cell lines after treatment with ATRA (all-trans-retinoic acid), a regulator of neural differentiation and proliferation. Ectopic overexpression of mir-125b stimulated glioma cell proliferation, partially recovering the cell growth inhibition induced by ATRA treatment, while mir-125b knockdown promoted ATRA-mediated cell apoptosis. Furthermore, Bmf was identified as a direct target of miR-125b, and they are inversely correlated [77]. Bmf interacts with the prosurvival Bcl-2 proteins, regulating cellular apoptotic pathways [78]. Indeed, transfection with miR-125b increase BCL-2 levels in glioma cells, and expression of BCL-2 was significantly decreased when cells were transfected with miR-125b inhibitor. Thus, Bmf may play an important role in the process of miR-125b influencing cell apoptosis [77].
Recently, Cortez et al identified that mir-125a downregulated in glioblastomas and particulary in glioma stem cells CD133+. Overexpression of mir-125a inhibits invasion properties of glioblastomas, probably through a direct downregulation of podoplanin (PDPN), a membrane sialo-glycoprotein related to invasion and malignancy [79].
Kefas et al evidenced a feedback loop between miRNA-326 and Notch pathway, frequently deregulated in gliomas. Notch-1 knockdown induced mir-326 upregulation in glioma stem cells, and ectopic overexpression of mir-326 decreased Notch pathway members level and activity. Forced expression of mir-326 also inhibited cell proliferation, viability and invasiveness and induced apoptosis in both established and stem cell-like glioma lines, these effects are at least partially explained through downregulation of the Notch pathway. Moreover, it reduced tumorigenicity in mouse glioma xenografts. Together with the observation that mir-326 is downregulated in glioblastomas, these findings suggest that miR-326 is a potential tumor suppressor in glioma cells and that reversing Notch/miR-326 axis toward miR-326 prevalence appears to be a potential therapy for brain tumors [80].
Mir-26a up-regulated in high-grade gliomas. MiR-26a is a direct negative regulator of PTEN and significantly represses PTEN expression. MiR-26a overexpression was attributed to the amplification of miR-26a-2 locus (chromosome 12q), one of the two miR-26a loci present in the human genome (mir-26a-1 and miR-26a-2) and is correlated with monoallelic PTEN deletion. It was suggested a temporal sequence to the molecular evolution of miR-26a-amplified gliomas, with PTEN loss most likely preceding miR-26a-2 amplification and, concordantly, miR-26a overexpression in genetically engineered PTEN+/- mice precluded loss of their remaining PTEN allele. Amplification of mir-26a is likely to promote the silencing of the remaing PTEN transcript in PTEN+/- tumors, event analogous to a loss of heterozygosity. Furthermore, in a murine glioma model, PTEN repression mediated by overexpression of miR-26a, enhanced de novo tumor formation. Akt pathway activation and suppression of its negative regulator PTEN are particularly important and frequently occur in glioma development. These observations document a new epigenetic mechanism for PTEN regulation in gliomagenesis, further highlighting that dysregulation of Akt signaling is crucial to the glioma development and could be modulated through manipulation of miRNA expression [64]. Moreover, MiR-26a is a frequent target of the 12q13.3-14.1 amplicon that also contain CDK4 and CENTG1, two oncogenes that regulate the RB1 and PI3 kinase/AKT pathways, respectively. The presence of this amplification is negatively correlated to patient survival. PTEN, RB1, and MAP3K2/MEKK2 were detected as functional targets of mir-26a in glioblastoma. Ectopic overexpression of miR-26a increased GBM cell growth, decreased apoptosis and enhanced gliomagenesis in vivo. Thus, miR-26a decreases PTEN expression, activates AKT, and promotes tumor growth. Using human U87 GBM cells that lack functional PTEN, mir-26a overexpression increased cell growth and decreased apoptosis despite the absence of PTEN, most probably by downregulating RB1 and MAP3K2, a gene that encodes MEKK2[81]. MEKK2 is a mitogen-activated protein kinase kinase involved in JNK activation pathway that promote apoptosis in GBM cells [82].
Mir-10b is upregulated in glioma samples and glioma cell lines compared to non-neoplastic brain tissues. RhoC and urokinasetype plasminogen activator receptor (uPAR), which are known to contribute to glioma invasion and migration, were correlated with mir-10b expression in gliomas, probably via inhibition of the translation of the mRNA encoding homeobox D10 (HOXD10], which in turn represses the expression of these genes [83]. These results suggest that mir-10 could be involved in regulation of the invasion and migration abilities of gliomas.
Mir-15b was shown to be elevated in a panel of glioma cell lines. Transfection of glioma cells with miR-15b significantly increased G0/G1 cells and decreased the percentage of cells in S phase, while inhibition of mir-15b increased G0/G1 cell amount. Thus, miR-15b regulates cell cycle progression in glioma cells by targeting cell cycle-related factors as CCNE1 (cyclin E1], a validated downstream target of mir-15b [84].
Mir-146b, a miRNA found to be downregulated in human glioma tissue, could exert an antitumor activity by reducing the expression of a matrix metalloproteinase gene, MMP16, one of its direct downstream targets. Indeed, transfection of U373 glioma cells with miR-146b precursor decreased tumor cell migration and invasion, while inhibition of miR-146b by LNA modified anti-miR-146b produced the opposite effect, without affecting cell proliferation [85].
Recent studies support a role of mir-296 in promoting angiogenesis in gliomas. When brain microvascular endothelial cells were co-cultured with U87 glioma cells or when vascular endothelial growth factor (VEGF) was added to cultured human brain microvascular endothelial cells, miR-296 was upregulated. Moreover, mir-296 levels were higher in endothelial cells isolated from human brain tumors compared to normal brain endothelial cells. Downregulation of miR-296 in endothelial cells resulted in the inhibition of morphologic characteristics associated with angiogenesis and reduced angiogenesis in glioma xenografts in vivo. This probably happens through the downregulation of the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a validated target of mir-296, thus leading to a reduced HGS-mediated degradation of the platelet derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor (VEGFR). This result points out an interesting feedback loop, where VEGF induces miR-296 expression, which in turn increases cell response to VEGF. Consequently, manipulating mir-296 expression, could enable to control a key step in cancer angiogenesis [86].
Mir-29b was found to be significantly downregulated in glioblastoma samples and cells and CD133+ tumor stem cells. Forced overexpression of this miRNA inhibited invasion and proliferation and induced apoptosis in glioblastoma cells. Mir-29b as well mir-125a directly targets podoplanin (PDPN), a putative marker of neural stem cells, related to invasion and malignancy in glioblastoma [13].
MiR-17-92 cluster, located within the locus 13q31-q32, encloses five miRNAs (miR-92-1, miR-19a, miR-20a, miR-19b, miR-17-5p), which have been frequently involved in tumorigenesis [87]. This cluster was detected to be amplified in glioblastoma samples. Furthermore, expression of miR-17-92 cluster is downregulated upon induction of differentiation of GBM spheroid cultures by using ATRA. Induction of differentiation leads to deregulation of most of the key pathways associated with self-renewal such as insulin-like growth factor 1 signaling, vitamin D receptor/retinoic acid X receptor activation, Wnt/β-catenin signaling and retinoic acid receptor activation. Inhibition of miR-17-92 cluster leads to decreased cell viability and decreased cell proliferation probably through a de-repression of CDKN1, E2F1, PTEN and CTGF which are upregulated. Particularly, connective tissue growth factor (CTGF) gene was shown to be a direct target of miR-17-92 in glioblastoma spheroids by luciferase reporter assays [88]. CTGF binds vascular endothelial growth factor (VEGFA), which is a central mediator of angiogenesis [89], and inhibits VEGFA-induced angiogenesis [90]. Conversely, VEGFA was shown to inhibit MiR-17-92 [91], thus explaining the concomitant downregulation of VEGFA and miR-17-92 upon induction of differentiation. In conclusion, the interaction between CTGF, VEGFA and miR-17-92 might have a key role in gliomagenesis by targeting multiple regulatory pathways [88].
Let-7 expression is not downregulated in human glioblastoma tissues and cell lines, let-7 ectopic overexpression by transfection on U251 and U87 human glioblastoma cells, reduced in vitro proliferation and migration and also in vivo tumor growth after xenotransplantation into nude mice. Furthermore, let-7 miRNA reduced the expression of total RAS, N-RAS, and K-RAS in glioblastoma cells [92]. These results suggest that let-7 miRNA is able to impair glioblastoma growth and cellular migration via RAS inhibition.
Glioma stem cells (GSCs) have been recently identified [93]. They express common neural stem cell (NSC) markers (CD133, Nestin, Musashi, and Sox2) and display multiple-lineage differentiation potential and a greater tumorigenic activity in rodent xenografts. GSCs also show an increased angiogenic potential through a higher expression of vascular endothelial growth factor (VEGF). GSCs are strongly resistant to radiation [94]and chemotherapy [95].
A recent study by Lavon et al showed a similar expression pattern of mir-21, mir-124, and mir-137 in gliomas and stem cells. Among them we mention the miR-17 family cluster that contains 3 miRNAs upregulated in gliomas and NPCs (mir-17-92, mir-106b-25, mir-106a); the mir-183-182 cluster, also upregulated in gliomas and NPCs; the large 7+46 bipartite miRNA cluster on chromosome 14, as most of miRNAs located within this region have been shown to be downregulated in the same samples [96]. This latter cluster is located within a region which shows a frequent loss of heterozygosity in glioblastomas [97]. Finally, mir302-367 cluster on chromosome 4q25 and mir371-373 cluster on chromosome 19q13, are upregulated in gliomas and NPCs. These data confirm the hypothesis that brain cancers arise from multipotent GSCs, thus explaining the phenotypic heterogeneity of tumors.
Many groups investigated the role of miRNAs in GSCs so far: in a first report Silber et al found that mir-124 and mir-137 are reduced in grade III and IV gliomas compared to normal brain. Besides, transfection of these two miRNAs in neural stem cells and glioma cancer cell lines leads to induction of cellular markers of differentiation, G1 cell cycle arrest and reductions of CDK6, thus indicating a hypothetical tumor suppressor role of micro-124 and microRNA-137 in GSCs [29]. As previously mentioned in this review, mir-128 was identified as a negative regulator of glioma self renewal when ectopically overexpressed on human glioma neurosphere cultures [58].
A recent analysis showed that mir-451 is significantly downregulated in CD133+cells. Transfection of miR-451 inhibits proliferation and neurosphere formation in GSCs, highlighting that it can act as a tumor suppressor: two target sites for SMAD protein in the upstream promoter region of miR-451 have been found, leading to draw the conclusion that this miRNA is activated by SMAD pathway. Transfection of SMAD in GBM cells inhibited their growth, suggesting it could induce differentiation of glioma CD133+stem cells through up-regulation of miR-451, thus reducing their tumorigenicity [98].In conclusion, a stable and selective delivery of these miRNAs to GSCs could represent a great advance for brain tumor therapy.
MiR -125b is required for stem cell fission, allowing them to bypass the G1/S checkpoint and making them insensitive to chemotherapy [99]. It has been found to be significantly downregulated in CD133+glioma stem cells compared to CD133-ones, leading to the hypothesis that it may be involved in cell differentiation. As expected, transfection of mir-125b to CD133+cells, decreased the number of proliferating cells and induced G0-G1 arrest: this effect occurs through a mir-125b-dependent downregulation of CDC25A and CDK6, two cell cycle regulatory proteins [47]. Moreover, miR-29b and miR-125a, are under-expressed in glioblastoma CD133+cells compared with their counterpart CD133-cells (see above), suggesting a potential role for these microRNAs in regulation of signaling pathways related to maintenance of stem cell properties and self-renewal of cancer cells [79].
Some miRNAs could have a role in the regulation of key pathways of GSCs. We already mentioned above the existence of a regulatory feedback loop between the tumor suppressor mir-326 and Notch pathway, shifted towards a prevalence of Notch activity in brain tumor cells and GSCs [80] Recently, pyruvate kinase type M2 (PKM2) has been identified as a putative target of mir-326, as levels of PKM2 and mir-326 are inversely correlated in glioma cells. PKM2, highly expressed on cancer cells and GSCs, plays the role of mediator in mir-326 metabolic effects: experimental knockdown of this molecule led to an impairment of glioma invasiveness and clonogenicity and decreased ATP levels, suggesting that PKM2 could represent a valid target for glioma therapy [100].
Mir-34a downregulated in gliomas and GSCs. When transfected into GSCs, it decreased the expression of the stem cell markers CD133 and nestin and caused an higher immunostaining for astrocytes and oligodendrocytes markers, besides modestly inhibiting several malignancy end-points (migration, survival, proliferation, cell cycle progression). Remarkably, miR-34a levels in glioma stem cells are significantly lower than in differentiated wild-type p53 glioma cell lines, suggesting that restoration of miR-34a expression for therapeutic purposes could achieve strong anti-tumor effects not only by targeting differentiated glioma cells, but also by inducing glioma stem cell differentiation [56].
Medulloblastoma (MB) is the most common brain malignancy observed in childhood (WHO grade IV) [101]. Ferretti et al were the first showed that miRNAs profiles is different between primary medulloblastomas and normal cerebellar controls. These aberrant miRNAs have a potentially role in MB development: e.g., let-7 microRNAs has been shown to inhibit Ras oncogene expression [102], reported to be a key factor for MB metastatic behavior [103]; miR-17-92 cluster cooperates with myc, frequently overexpressed in MB, to induce neoplastic transformation [104]; miR-9 and miR-125a, both downregulated in MB, are involved in cell proliferation and, when transfected into MB cells, promote apoptosis and impair anchorage-independent growth by downregulating the truncated isoform of the neurotropin receptor TrkC (t-TrkC). T-TrkC expression levels are higher in MB, inversely correlate with miR-9 and miR-125a levels and are responsible of enhanced cell proliferation and worse prognosis. Mir-9 and mir-124 have a common molecular target, REST/NRSF (RE1 silencing transcription factor/neuron-restrictive silencer factor) complex [105]. These observations suggest that Mir-9 and mir-124 could play an important role in cerebellar tumorigenesis as REST inactivation has been reported to inhibit tumor growth [106] and is overexpressed in many MBs [107]. Consequently, a REST/mir-124 axis shifted towards a prevalence of REST activity, could block neuronal differen-tiation and promote neoplastic transformation [108].
Mir-124 also modulates medulloblastoma growth by targeting CDK6, a key pro-proliferative factor overexpressed in 30% of medulloblastomas, which represents an adverse prognostic marker for clinical outcome [109]. The role of mir-124 in MB development was later confirmed by Li et al who additionally demonstrated that ectopic expression of mir-124 in medulloblastoma cell lines inhibits cell growth by directly targeting SLC16A1, a protein upregulated in MBs, that serve as a carrier to export lactic acid extracellularly, thus maintaining homeostasis of tumor cells, where aerobic glycolysis is known to be accelerated [110].At the same time, downregulation of miR-218 could account for an overexpression of the pro-oncogene EGFR, which activates MAPK pathway and CTNND2, which in turn encodes the gene of β-catenin that activates the APC/Wnt signal transduction pathway and leads to tumor growth [111].
Hedgehog (Hh) patched (Ptch1) signalling pathway is a key regulator of the development of cerebellar granule cell progenitors and its constitutive activation makes cells susceptible of malignant transformation into medulloblastoma [112]. Hh is a secreted protein that binds to the transmembrane receptor Ptch1 transducing an intracellular signal through the protooncogene Smoothened (Smo) to the downstream transcription factors Gli1, Gli2, and Gli3 [113].
Ferretti et al found that miR-125b, miR-326 and miR-324-5p target and repress activator downstream components of the Hh signalling pathway (Smo and Gli1); consistent with these findings, 50% of MBs showed a downregulation of these miRNAs together with an overexpression of Gli1. Deletion of chromosome 17p, the most frequent mutation in medulloblastoma, could account for several genetic defects, including miR-324-5p, p53 and HIC1 tumor suppressors loss, which cooperate in Hh-dependent tumorigenesis: loss of these genes, together with other molecular events, contribute to a persistent hyperactivation of Hh signalling during cerebellar granule cell progenitors development, leading to higher proliferative activity and susceptibility to malignant MBs [114].
Recently, Hedgehog signalling pathway has been identified as a target of miR-17-92 cluster. Indeed, miRNAs from the miR-17-92 cluster are specifically overexpressed in mouse MB models with specific initiating mutations in Ptch1 and in human MB subgroups with an activated SHH signaling pathway. To evaluate its oncogenic potential, miR-17-92 was retrovirally transduced into mouse granule neuron progenitors cells (GNPs) before orthotopic transplantation into immunocompromised mice. Interestingly, only cells with an SHH signaling defect (Ptc+/-) developed MBs. Tumor cells from this model exhibited markers of activated SHH signaling as elevated Math1 and Gli1 mRNA levels, and lost expression of the wild-type Ptc allele, reinforcing the hypothesis of a functional link between the SHH pathway and this miRNA cluster [115]. Some studies showed that miR-17-92 is most highly expressed in SHH-driven medulloblastomas, and higher levels of miR-17-92 are also related to an overexpression of the oncogene MYC. Moreover, transfection of miR-17-92 maintains mouse CGNP cells in a proliferative state in the absence of SHH, and synergizes with SHH to promote cell growth, while treatment of the same cells with SHH results in upregulation of miR-17-92, confirming that this cooperation is crucial in MB tumorigenesis [116].
Notch pathway plays a key role in regulating granule-cell progenitor differentiation and an increased copy number of Notch-2 was detected in 15% of medulloblastomas [117]. Expression of the downstream effector of Notch, HES1, normally declines during neuronal differentiation, while persistent activation of this factor prevents differentiation of precursor cells [118]. Garzia et al identified mir-199-5p as capable of directly targeting and repress expression of HES1 in MB cell lines. Ectopic expression of this miRNA reduced MB cell proliferation, population expressing stem cell marker CD133 and xenograft tumor growth in mouse models. Their studies also suggest that the documented downregulation of miR-199b-5p in metastatic tumors may be related to epigenetic silencing [119].
Venkataraman et al recently showed that several miRNAs known to be involved in CNS development, are downregulated in medulloblastoma cell line cultures and tissues. A specific one, miR-128a, is able to decrease tumor growth and proliferation if ectopically re-expressed on MB cells. Bmi-1 has been identified as a putative target of miR-128a, a critical factor in cerebellar development frequently upregulated in MBs. MiR-128a seems to normally downregulate Bmi-1 oncogene, leading to increased levels of p16, a cell cycle inhibitor. Furthermore, Bmi-1 could be involved in regulating reactive oxygen species by decreasing superoxide generation, thus leading to a lower redox state in cancer stem cells [120]; this event is known to be partially responsible of tumor resistance against common therapies [121].
Finally, mir-34a appeared to be a tumor suppressor gene also in medulloblastomas other than in glioblastomas. Indeed, transient transfection of miR-34a into medulloblastoma cell lines, strongly inhibited cell proliferation, cell cycle progression, cell survival and cell invasion most probably through a direct inhibition of c-Met [56].
Pituitary adenomas are benign and frequent neoplasms, accounting for about 15% of primary intracranial tumors [122]. Bottoni et al found that 30 miRNAs are differentially expressed between normal pituitary gland and pituitary adenomas. Furthermore, their expression is inversely correlated with tumor size. They probably act through a negative regulation of RARS (arginyl-tRNA synthetase), which is in turn upregulated in pituitary adenomas and modulates the expression of factors influencing pituitary tumor growth [123]. Mir-15a and mir-16-1 are located within chromosome region 13q14. Interestingly, loss of 13q region of chromosome 13 was frequently detected in pituitary tumors, confirming that this region likely contains tumor suppressor genes [124].
These findings support the hypothesis that miRNA-16-1 plays a key role in tumor growth [195], most probably by interacting with BCL2 [125], which is overexpressed in about one third of pituitary adenomas [126]. MiRNAome could also function as a signature for specific histotypes of pituitary adenomas. Overexpression of miR-23a, miR-23b, and miR-24-2 and lower expression of mir-26b are more typical of GH-secreting and PRL-secreting adenomas, differentiating them from non-functioning adenomas (NFA), characterized by mir-26 upregulation and miRNA-24-2 downregulation. Moreover, NFA express higher concentration of miR-137 and lower of miR-127, miR-129, miR-203, and miR-134 compared to GH-secreting adenomas. Finally, ACTH-secreting adenomas are defined by a strong expression of mir-30 cluster (miR-30a, miR-30b, miR-30c, and miR-30d), supporting the hypothesis that miRNAs could be useful diagnostic markers to distinguish pituitary tumor histotypes [127].More recently, Amaral et al evaluated the expression of microRNAs in ACTH-secreting pituitary tumors: they found that let-7a, miR-21, miR-141, miR-143, miR-145, and miR-150, besides miR-15 and miR-16, are downregulated in corticotropinomas compared to normal pituitary tissue and that a lower expression of mir-141 is linked to a better chance of remission after surgery [128]. Moreover, downregulation of Mir-143 could be involved in tumorigenesis by activating MAPK pathway via ERK5 [129].
Quian et al showed that let-7 expression is decreased in more than one third of pituitary adenomas and is related to higher tumor grade, thus it may act as a tumor suppressor. Levels of let-7 were found to be inversely correlated to HMGA2 expression [130], confirming some previous studies which showed that HMGA2 is repressed by let-7 [131]. High levels of HMGA2 have been detected in most types of pituitary adenomas [132] and they are significantly associated with tumor grade, extent of invasion, tumor size and Ki-67 proliferation index, probably through regulation of E-cadherin, E2F1, cyclin A, and p53 expression. Accordingly, let-7 may be useful as a novel anticancer agent in the future [130]. A recent study on the role of miRNAs in NFAs and GH-secreting pituitary tumors was performed by Butz et al, showing that a group of miRNAs (miR-128a, miR-155, and miR-516a-3p) targets the 3\'-untranslated region of Wee1, a nuclear protein kinase that acts as a tumor suppressor, and which was found to be significantly decreased in pituitary tumor samples [133].
Current evidence indicates that miRNA deregulation is common in human cancers. The discovery of miRNAs with oncogenic or tumor-suppressive function raises the possibility of exploiting these RNA molecules for therapeutic intervention and the development of novel therapies. The oncogenic miRNAs can be downregulated using antisense miRNA oligonucleotides (AMOs or antagomirs) or miRNA sponges[134,135], whereas tumor-suppressive miRNAs may be targeted by replacement with miRNA mimetics.
AMOs are synthetic oligonucleotides with sequence complementary either to mature miRNA or its precursor and have been widely used to inhibit miRNA activities in vitro and in vivo[136]. A number of chemical modifications to AMOs have been developed to improve specific binding to target miRNAs and to resist nuclease degradation. These include modifications at the 20-hydroxyl position (20-O-methyl (20OMe), 20-Omethoxyethyl(20MOE), 20-fluoro (20F) and locked nucleic acids (LNA)), on the phosphorothioate backbone, and conjugation with cholesterol [137]. Of these modifications,LNA shows high stability, strong affinity for target miRNAs and low toxicity in biological system and has emerged as a strong candidate for targeting miRNAs in vivo. One clinical trial using LNA anti-miRNA in treating human disease has been initiated recently. Using LNA antimiR-21, Corsten et al. demonstrated that miR-21 knockdown in glioma cells led to an increase of apoptotic cell death and a significant reduction of glioma growth in vivo, suggesting that miR-21 is a potential therapeutic target. They further showed that combination of miR-21 suppression and the cytotoxic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in enhanced caspase activity and synergistic killing of glioma cells in vitro. When these treated glioma cells were implanted intracranially into nude mice, tumor cells were completely eradicated after day 6 of implantation, compared to reduced tumor volume with either treatment alone. This finding suggests that miR-21 knockdown sensitizes glioma cells to apoptosis agent, and that the combined treatment is a promising approach for glioma elimination [138].
Angiogenesis is a process essential for malignant glioma growth. By co-culturing glioma cells with microvascular endothelial cells, Wurdinger et al. showed that glioma cells could induce changes of miRNA expression in endothelial cells, with miR-296 being significantly upregulated. Enhanced expression of miR-296 was also observed in endothelial cells treated with glioma-conditioned medium or proangiogenic growth factor VEGF or EGF, as well as in endothelial cells isolated from glioma samples. These results suggested a role for miR-296 in angiogenesis. It was further demonstrated that miR-296 contributed to angiogenesis by targeting hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), which controls the levels of growth factor receptors (VEGFR2 and PDGFRb) by directing these macromolecules to lysosomes for degradation. Importantly, intravenous administration of antimiR- 296 AMO significantly reduced angiogenesis in glioma xenografts in vivo[139]. These results suggest that inhibition of angiogenesis by targeting miR-296 in endothelial cells may represent an alternative approach in glioma therapy.
MiRNAs are an astonishing new class of gene regulators, and it had been demonstrated that these molecules play a crucial role in cancer development and progression in a variety of malignancies, including brain tumors. Most importantly, in a clinical context, there is first evidence that miRNAs might provide new options to improve diagnostics and therapy in the two most common malignant primary brain tumors in adults (glioblastoma) and children (medulloblastoma). In vitro and in vivo data suggest that miRNAs could be used to discriminate brain tumors from normal brain tissue, and to identify different astrocytoma grades. More important, clinico-pathological features seem to correlate with miRNA expression in these tumors. Furthermore, there is increasing evidence that miRNAs might help to generate targeted therapies and to overcome resistance to conventional anticancer strategies for example in glioblastomas. Of course, it has to be acknowledged at this stage that translation of these preliminary “in vitro data” into “hard clinical facts” is not feasible. But these findings provide a very promising basis for future studies to determine the effect of miRNA modulation on chemotherapy in “in vivo studies”. Although therapeutic delivery of miRNAs is still a developing field, and there is much more work to be done before these molecules can be securely applied in clinical settings, miRNA modulation may one day have a therapeutic application in patients. In summary, the presented data supports the enormous clinical potential of miRNAs in brain tumors, and mandate further intensive investigations in this field.
Supported by China National Natural Scientific Found (30901772).
Organoids are miniature 3D models of
Organoid formation from stem and progenitor cells. Following tissue dissociation, adult stem cell/ progenitor cells from normal or patients are isolated. Induced pluripotent stem cells (iPSCs), embryonic stem cells and adult stem cells are subjected to guided differentiation, followed by growing them on extracellular matrix in 3D culture system using specific culture media to begin organoid culture. The lower right section shows the different aspects of organoid commercialization, including molecular profiling of the collected cells, long term storage of cells with appropriate patient consent and information. These biobanked organoids can then be used for functional studies related to potential applications like disease modeling, therapeutic development, regenerative medicine, toxicology, and personalized medicine.
Tissue specific organoid bioenginnering and different basic biomedical and commercial applications.
Backed by some significant observations in the proliferative nature of adult tissue stem cells in 2009, the cardinal notion enveloping organoid technology is that stem cells have ingrained potential to self-assemble into 3D constructs with similitude with human organs [5]. As time rolled on, the modus operandi has been implemented to produce several human and murine organoids out of epithelial tissues of various organs such as the liver, intestine, kidney, skin [6, 7, 8, 9]. Another breakthrough entails the evolution of organoids obtained from induced pluripotent stem cells (iPSC), which can circumvent the toil to avail specific tissues like the heart or brain. This prodigious prospect, reinforced with genetic engineering, permits the mutational corrections in patient-derived iPSCs expediting differentiation to generate a specific type of cells [10, 11, 12, 13]. Scrupulous experimental manipulation while maintaining sensitive biological complexity allows organoid technology to bridge the gap between 2D cell culture and 3D models [14]. It has also proved to better simulate human physiology than animal models and has shown the promise to substitute animals in preclinical biology [15, 16].
As ratiocinated from the trends, there will be a steady increase in the demand for organoid technology in the following years [3, 16]. As per reports published in various media, around 20 companies are into business with this technology—their activities include biobanking, manufacturing, commercialization, the implication of robotics for the development of organoids, organoids on a chip, etc. Statistically, priorities are given to heart, brain, intestine, and kidney organoids [3]. Financial models adopted by these companies are—(i) venture capitals, (ii) partnerships (iii) direct collaboration with the originators. Routine use of animals for disease modeling has always been afflicted with stringent moral and ethical queries. Usage of embryonic stem cells has also faced stormy skirmishes regarding the moral status of the embryos. Likewise, the moral and legal status of the organoids has been called in regulatory questions, to mention a few—ownership, consent, IP rights, safety, commercialization, etc. Utilization of ‘matrigel’ (extracellular matrix obtained from animals) has raised some safety concerns regarding compatibility with the human system. The debate revolving around the exchange or donation of human tissue as a commodity is still ongoing. To resolve this, few regulations should be declared and accepted by the global intellect [17]. Quibbles for intellectual property (IP) generation with human tissue should cease to persist, showing proper dignity and preserving the donor\'s rights. Consent should become a requirement avoiding de-identification of the donor [18, 19, 20, 21]. In the present chapter, we shall highlight the usage of the organoid, organoid cell atlas, followed by shedding light on the commercialization aspect of the technology and future directionality.
Researchers traditionally used
With the advent of tumor organoid culture, patient-derived tumor organoids (PDTOs) have become popular tools to study molecular tumorigenesis, understand tumor heterogenity, predict drug responses, immunotherapy, and precision cancer therapy. At the moment, several tumor organoid biobanks have been developed catering to a variety of cancer types, including lung [31], breast [32], gut [33], and brain [34], liver [35], colorectal [36], pancreas [37], prostate [38], and ovary [39]. The role of tumor immune microenvironments (TIME) is significant in improving cancer immunotherapies, and PDTOs have started playing a crucial role in modeling the tumor-immune landscape. PDTO-based TIME studies can help evaluate immunotherapies such as checkpoint inhibition and adoptive T-cell treatment [4]. Thus, optimizing the tumor organoid culture method is critical for developing organoid-guided customized cancer immunotherapy [40].
Human organoids are suited for genetic modification and customization and bridge the gap between fundamental research and clinical practice. This technique has aided oncology, biological, pharmacological, regenerative, and personalized medicine studies [1]. Despite the initial advances, the technology is still nascent and is expected to be employed in various applications such as developmental and stem cell biology, toxicology, drug discovery, personalized medicine, disease modeling, immune interaction, and regenerative modeling (Figure 2). Healthy human organoids from different tissues may be utilized to test comparative therapeutic toxicities in combination with disease-related organoids. Cardiac organoids, liver organoids, kidney organoids, and other organoids are now used to dictate intolerable adverse effects, such as hepatotoxicity, cardiotoxicity, nephrotoxicity, and other tissue toxicity [41]. Patient-specific relevant organotypic models will go a long way in reframing basic findings, testing innovative ideas in 3D, and validating crucial data without sacrificing animal life for science. This aspect is dealt with in other publications [42, 43].
The lack of a physiologically relevant model system has delayed the therapeutic development process, and many candidates have failed in clinical trials [4]. Cancer organoids are near-physiological replicas of their parent tumors and bridge the gap between drug screening and clinical trials. Organoids have been utilized to examine personalized cancer patient responses in several research [32, 44, 45]. It may also be utilized to look at the epigenetic and genetic changes that cause drug resistance [46]. Tumor organoids can accurately predict chemotherapeutic response and resistance for certain drugs in some cancers [47, 48]. Finding the right therapeutic combination can be a challenge, and tumor organoids can help solve this dilemma and can make personalized medicine a reality [49]. The advances in genetic engineering technologies like CRISPR-Cas9 are implemented on organoids further to confirm medication sensitivity to specific mutations [50]. Organoids may also be used for pharmacokinetic research, critical in drug development. Results suggest that drug-transporters, their efflux transport functions, drug-metabolism can be efficiently studied using organoids [51]. In addition to cancer biobanks, organoids have a significant role to play in immunotherapy, a kind of cancer treatment in which the patient\'s immune system is used to eliminate tumor cells [52]. Organoid-based models are explored to study the effect of tumor-immune cell interaction using a coculture system with both components [53].
Organoids holds promise as a propitious platform in biomedical research and applications for many decades to come. Human organoids currently have a few limitations that require to be circumvented to appreciate their full potential. Some technical and conceptual limitations may be addressed using single-cell sequencing and spatial profiling. Single-cell transcriptome/ epigenome sequencing and spatial profiling can provide a thorough idea about the composition of cells and the state of cells present within the organoids, which may help develop organoids as futuristic models of human biology. In combination with the Human organoids and single-cell technology, a pilot project has been launched within the Human Cell Atlas (HCA) as a “Biological network” (https://www.humancellatlas.org/euh2020/) [54]. HCA is a revolutionary global collaborative initiative aiming at advancing biomedical research opportunities and therapy using single-cell technologies (https://hca-organoid.eu/). This pilot project, under HCA, focuses on the single-cell characterization of organoids and other complex
It is one of the six pilot projects funded by the European Union (EU) Horizon 2020 Framework Programme, which will be helpful in developing the first version of the Organoid Cell Atlas, which may be used as a nucleus for a broader, collaborative, global initiative. The HCA-Organoid association has eight partner Institutions, including EMBL’s European Bioinformatics Institute institutions having experts in organoid technology, single-cell profiling, advanced imaging, and bioinformatics from Austria, Germany, the Netherlands, and Switzerland, and received €5 million by EU funding, as a part of the European contribution to the HCA project. Currently, the project mainly focuses on generating single-cell transcriptome, epigenome maps, and detailed imaging data in a selection of human organoids. The initial objective of the funded project is to derive and characterize two organoids, colon and brain, from 100 whole-genome-sequence individuals each, to have a record of normal population variation and have a reference for disease-centric research [56].
The colon and brain organoids were one of the first organs to which organoids were demonstrated, so comparatively, more advanced protocols for the two are available with HCA [2, 23]. Apart from this, the colon organoids are derived from adult stem cells while the brain is from the iPSCs, thus spanning the two primary sources of organoid derivation. Both of them have primarily been used for disease-centric studies. If the single-cell characterization of these organoids is done for many individuals, this can help facilitate various biomedical applications. Beyond the initial target, most of the data information in the project is generalized in a way to be applicable to various other types of human organoids. The HCA has also spoken about the possibility that they can collaborate with other institutes for different projects, which can pursue systematic single-cell profiling in other types of human organoids, to explore the possibility of interrelation with the Organoid Cell Atlas [56].
The main aim of the EU H2020 HCA-Organoid project is to build an Organoid cell atlas portal that may be equipped with the computational infrastructure and a web-based front end that makes the data easily accessible and analyzed. Some of the organoid-specific features that have been focused on while developing an Organoid portal include the interactive exploration of human organoid data, data-driven selection of organoids for functional experiments, and comparison of disease-specific organoids against reference collections of normal organoids.
This portal also focuses on providing the data of the corresponding primary tissues available in the HCA and also will work on showing interactive mappings between single-cell profiles of human organoids. These may be achieved using the algorithms that enable cell-cell alignments between these datasets. This portal is supposed to facilitate the use of organoids in biomedical experiments and encourage the use of organoids as models in various experiments like precision medicine, drug development, disease modeling, etc. Mapping and data integration may detect normal variation between individuals in an interactive manner showcasing organoid as the capable model for the corresponding variation in primary tissues. The analysis and interpretations of disturbances in the human organoids related to the primary tissues will be performed using cell-cell alignments [56].
A set of strategies have been laid to develop the atlas to be most productive and of high quality. Initially, it was thought to invest in validation and standardization for organoid-related research. Later, a contribution towards the HCA to establish community standards and software infrastructure for data processing and data annotation was strategized. Then the development and validation of computational methods for the comparison of cells between organoids and corresponding primary tissues and their flexible alignments were implemented. Finally, the implementation of interactive visualization tools that helps in establishing user-friendly quality control and exploratory analysis of single-cell organoid datasets contributed to the Organoid Cell Atlas [55].
With the development and successful commercialization of organoids, the most demanded sector in treating patients and pre-clinical trials in pharmaceutical industries will give a slanting graph in the market in the future [57]. In this present era, many specific and most suitable techniques have been developing over the years for organoid research, leading to competition among industries worldwide. The annual cost of treating brain diseases in Europe is $798 billion, and globally it amounts to $3 trillion. Over 90% of novel drugs that are being developed for brain diseases fail during the developmental process, which further reinforces the scope and opportunities for organoids [2]. The development of the living human brain (LHB) enables culturing of human-derived brain organoids from the cells of any individual; the University of Helsinki provides a new technical idea for preclinical trials of drugs in this area. Helsinki Innovation Services Ltd (HIS) supports the commercialization projects of the University of Helsinki from the funding application stage to completion.
Many startups such as XILIS, CELLESCE, SYSTEM1 BIOSCIENCES, 3DYNAMICS, PATH BIOANALYTICS, KNOWN MEDICINE, CYPRE, DYNOMICS are currently working in the domain of organoid technology. XILIS is developing a patient-derived miniature organoid technology to upgrade precision medicine and pharmaceutical drug discovery; their needed materials lead to 30× speed, 50× throughput, and 300× cost-saving (Hans Clever includes in the founding team of XILIS). CELLESCE is a UK-based startup that invented a bio-processing technology intended to grow and expand organoids in drug discovery and regenerative medicine. $25 million of Series A venture funding is raised in SYSTEM1 BIOSCIENCES incorporation with Charles River Ventures and Pfizer Ventures, upgrading neuro drug discovery through the combined action of human brain models, scaled biology, and machine learning to interpret brain disease from genetics to neural computation. Also, KNOWN MEDICINE raised a total of $2.4 M from Khosla, Cota Capital, and Y-Combinator, offering cutting-edge biology research and the latest AI techniques, giving oncologists an easy spot for treating patient\'s tumors with the best suitable drug. PATH BIOANALYTICS does bioanalysis of Phenotypic drug discovery and development. CYPRE is developing a tumor model platform intended for transformative 3D cellular research and clinical testing of cancer patients with seed funding from Hemi Ventures and others. DYNOMICS received $500K in pre-seed funding from Boost VC. The details of the organoid-specific startups are presented in Table 1. These different startups contribute a vast platform to save millions of people’s life [58]. Several companies are also operational in the tumor organoid domain, like Charles River and CROWNBio.
Company name | Country | Application area |
---|---|---|
XILIS INC. | Durham, North Carolina, USA | Micro-organospheres |
CELLESCE | Wales, UK | Patient-derived organoids (PDO) |
SYSTEM 1 BIOSCIENCES | San Francisco, CA, USA | Brain organoids |
3DNAMICS | Baltimore, Maryland, USA | Brain and liver organoids |
PATH BIOANALYTICS | North Carolina, USA | PDO for precision medicine |
KNOWN MEDICINE | Salt Lake City, Utah, USA | Patient-specific organoids for cancer drug development |
CYPRE | San Francisco, CA, USA | 3D tumor model |
DYNOMICS INC. | San Francisco, CA, USA | Human cardiac organoid |
CHARLES RIVER | Wilmington, MA, USA | Tumor organoid |
CROWNBio | San Diego, CA, USA | Tumor organoid |
The current leaders in the commercialization of organoids, their geographical location, and application areas.
Despite using pre-clinical trials in animal models, concerns are raised about whether the animals will be extinct if used over the years. This will lead to the depletion of species and a significant effect on the ecosystem. Moreover, they may lead to harmful effects on the environment once mutated and released. The Animal Welfare Act of 1970 was implemented in the United States and set standards for animal use and care in research. Three principles of the Act are (1) experiments must be proven necessary for instruction or to save or prolong human life, (2) animals must be appropriately anesthetized, (3) animals must be killed as soon as the experiment is over. Much to our intrigue, different types of organoids such as kidney organoids, lung organoids, liver organoids, intestine organoids, brain organoids, etc., can substitute such animal models in preclinical trials for drug discovery and precision medicine. Even self-organ plantation may occur with the continuous development of organoids.
Globally, the expenses of organ transplantation and post-transplantation maintenance treatment are quite expensive but varies according to variables such as geographical location, medical facility, transplant organ type, and access to insurance coverage [59]. Private hospitals in India now charge around INR 10 lakh to INR 30 lakh for a heart transplant, while in USA, the charges can be very high ~$1,664,800.00 (https://www.statista.com/statistics/808471/organ-transplantation-costs-us/). While the cost of a kidney transplant goes from INR 5 lakh to INR 20 lakh, while in USA it can be around ~$442,500.00. The cost of a liver transplant runs from INR 15 lakh to INR 35 lakh, and ~$878,400.00 in the USA. The eventuality of the unaffordability of such expensive treatment modalities led to the demise of a multitude. Furthermore, organ transplants from other donors are sometimes associated with organ rejection and the onset of auto-immune disease. It is not only the high cost of transplant but also the availability and transport of organs is a major issue in many countries. Though the number of donors have increased but the needs have also reached new heights [59]. One-third of all organ transplants fail due to rejection due to multiple reasons including HLA mismatch and alloantibodies [60]. While modern medicine has halted acute rejection but chronic rejection is a major challenge. The organoid technology may provide a realistic patform to design transplantable tissues in a dish thereby catering to the transplant problem in the near future as current limitation prevent organoids from meeting these expectations.
Another recent scientific area where organoids showed great potential was during the COVID-19 pandemic caused by the SARS-CoV-2 coronavirus. SARS-CoV-2 causes respiratory illness and multi-organ dysfunction. Scientists were scrambling to test experimental COVID-19 systemic medicines. Organoids were used to study the adverse effects of SARS-CoV-2 infection on human tissues and for the investigation of prospective therapeutic approaches. With the recent work on mini lungs organoids, a few of the drugs stemmed from the infection of the organoid models, representing a handful of possible treatments for COVID-19 [26]. Scientists must still develop methods to produce more complicated systems, such immune cells and blood arteries, to fully harness the technology [26]. Scientists must also find a way to swiftly and inexpensively produce thousands of identical organoids. Bioprinting is a potential new fast prototyping method that prints cells and accompanying matrices in 3D. Organoid bioprinting uses hydrogel-based bioinks to deposit various cell types that stimulate physiological signaling and can help commercialize the platform faster [61].
According to a study published by Fior Markets, the global organoids, and spheroids market is predicted to increase from USD 502.92 million in 2019 to USD 2794.79 million in 2027, with a CAGR of 23.91% from 2020 to 2027 [57, 62]. Till now, 19 companies are having an interest in organoid commercialization. Some of them are Thermo Fisher Scientific (Waltham, MA, USA), Merck (Kenilworth, NJ, USA), Corning (Corning, NY, USA), STEMCELL Technologies (Vancouver, Canada), Lonza (Basel, Switzerland), Prellis Biologics (San Francisco, CA, USA), Amsbio (Abingdon, UK), Cellesce (Cardiff, UK), DefiniGEN (Cambridge, UK), OcellO B.V. (Leiden, Netherlands), HUB Organoid Technology (Utrecht, Netherlands), 3Dnamics Inc. (Baltimore, MD, USA), Organoid Therapeutics (Pittsburgh, PA, USA), InSphero (Schlieren, Switzerland), etc. Organome (Baltimore, USA) and HUB (Hubrecht Organoid Technology) are dedicated to organoid biobanking, and other companies aim at manufacturing, organoid marketing, other related technologies. SUN Biosciences (Lausanne, Switzerland) and System1 Biosciences (San Francisco, CA, USA) use robotic automation tools for organoid generation. The semi-automated process enabled researchers to make retinal organoid production and selection faster using the algorithm. The MIMETAS (Leiden, Netherlands)—the organ-on-a-chip company, offers the second-best cell-based model after humans, using human cells growing in three-dimensional structures called Mimetas’OrganoPlates (microfluidics-based culture plates allowing culturing and screening of a range of organ and tissue models), which are affordable and available for nonspecialized end-users. With a consumption market share of about 46% in 2019, North America is the most important consumer of organoids, with Europe in second place. Key manufacturers of the global organoids market are Thermo Fischer Scientific, Merck, and Corning. The top three players took up a market share of about 75% in 2019. Byers of the report can access verified and reliable market forecasts, including those for the overall size of the global organoids’ market in terms of revenue. The Organoids’ market is segmented into 3D Organoid Culture and Biochemical Cues. In the case of Organoids application, the leading players are Biopharmaceutical Companies, Contract Research Organizations, Academics, and Research Institutes. The regional analysis covers North America (USA, Canada, and Mexico), Europe (Germany, France, UK, Russia, and Italy), Asia-Pacific (China, Japan, Korea, India, and Southeast Asia), South America (Brazil, Argentina, Columbia, etc.), Middle East and Africa (Saudi Arabia, UAE, Egypt, Nigeria, and South Africa) and predicts an upsurge in the usage of organoid technology across the globe in future.
Organoid Biobank is like a commercial bank with a similar modus operandi. In organoid biobank, collected samples from different sources such as stem cells, primary tissues, and biopsies were made into organoids and stored. The organoid samples can be taken from a healthy individual and a patient. These stored organoids are ready to use for different purposes. Organoid biobanks manage the database of organoids and a registry with all the patient details. These stored organoids can be tracked and used for wireless phenotyping with the help of radio-frequency identification (RFID) ultracompact chips inserted within them. The organoid biobanks store and transport organoids using liquid nitrogen.
The regulatory rules for organoid research use and commercialization are not very well laid in many countries and needs an update. The scientific, ethical, and regulatory problems related to organoid research are subject to extensive regulatory scrutiny and controlled partly by federal laws and state laws in the US and pertain to International laws in case of foreign collaborations. United States Department of Health and Human Services (HHS) and the Food and Drug Administration (FDA) regulations are implemented in organoid research [63, 64, 65]. Organoid research is subject to the Institutional Review Board (IRB) approvals. Many institutions have Embryonic Stem Cell Research Oversight (ESCRO) or Stem Cell Research Oversight (SCRO) committees that oversee research utilizing human ESCs or iPSCs. These committees are outlined in the National Academies Guidelines for Human Embryonic Stem Cell Research and help evaluate the scientific and ethical aspects. The special committees assess the current state of research, weigh the advantages and hazards, address related ethical problems, including informed consent from the donor, and evaluate suitable supervision methods as per the federal guidelines [63].
While in Europe, organoids research must be approved by the Research Ethics Committee (REC) as per the guidelines laid down by the European Medicines Agency (EMA). Different European countries also have their individual regulatory agencies. In India, the regulatory board is the Institutional Committee for Stem Cell Research (IC-SCR). At the same time, in China, the measures for Ethical Review of Biomedical Research Involving Human Subjects and Ethical Guidelines for Human Embryonic Stem Cell Research, are used as guidelines [66]. The manufacturing process of organoids for commercial purpose must fulfill the same standards as other pharmaceutical drugs, following good manufacturing practices (GMP) [64, 65]. Additional hurdles exist at the convergence of organoid technology and commercial clinical use, global rules relating to the heterogeneity of approved quality standards, privacy laws and data protection, patent laws and identifying ownership. As a positive step in this direction, the International Society for Stem Cell Research (ISSCR) has set new stem cell research guidelines in 2021 [67, 68, 69]. It is expected that organoid-based biotechnology innovations would need updated global regulatory guidelines and governance in the future.
Organoid technology and biobanking have grown in recent years, paralleling the expansion of stem cell and organoid research. However, ethical, moral, and legal existential questions persist. One crucial aspect is establishing globally recognized consent procedures for research and clinical organoid biobanking protocol. Also, the ethical and moral implications for clinicians while using organ mimics should be called into question. Patenting concerns are bound to arise when organoids are distributed over the world. Collaborations between the public and commercial sectors may lead to data exchange, entitlement sharing, etc. Confidentiality decorum and global patenting rules for organoid rights are therefore evolving. Even the iPSC-based organoids bring new concerns around permission, commercialization, ownership, IP rights, safety, and marketing [70]. The proliferation of big data, genomics, biobanking, and the globalization of the biotechnology sector has complicated the task of setting universal ethical standards. The primary ethical consideration about organoids is who owns them and whether small organ mimics (similar to organs) can be traded? With the advances in organoid technology, where do we draw lines to differentiate between organoids and tissues/organs? Sample de-identification, donor consenting, and licensing rights need to be better defined, especially concerning organoid commercialization. Addressing these bottlenecks may allow quicker commercial application for drug discovery, disease modeling, and research and development.
In the upcoming years, the designed pilot project focuses on substituting animal or human models with the organoid model by encouraging better research and accessibility of organoids. The HCA has been encouraging this shift of models by establishing a reference map of the entire human cells; which will be the first molecular picture of a human that will help the researchers to functionally dissect and systematically analyze human biological systems that would lead to a better exploration of organoids as a model. The initial version of the organoid cell atlas is planned to be established by the upcoming year that will be practically useful and open to advancements. These are thought to help maximize the impact of the project to become helpful in the field of basic biology and biomedical applications. To broader the reach, the single-cell profiles will be made public as soon as possible following HCA\'s ethical, social, and legal guidelines. Finally, the Organoid Cell Atlas Portal will be made “into a public, sustainable and widely used infrastructure for finding, accessing, analyzing and interpreting single-cell data from human organoids.” The goal of the Organoid Cell Atlas is to make advancements in the biomedical field and develop various regenerative therapies by encouraging and accelerating disease-centric research of rare genetic diseases, precision oncology, or other complex diseases that are yet less understood. So, to achieve all of their objectives, they have made the portal open to create an inclusive research environment that would help in collaborating with a broad range of researchers interested in the field, which would later lead to extremely well-defined growth in the field of organoids.
M.K.P. acknowledges S. Dubinett, B. Gomperts, and V. Hartenstein for providing constant support and mentoring.
The authors declare no conflict of interest.
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\n\nInspect your order carefully when it arrives. Any problems should be immediately reported to orders@intechopen.com.
\n\nPrint copies of our publications are most often purchased by universities, libraries, institutions and academia personnel, hence increasing the visibility and outreach of our authors' published work among science communities and institutions.
\n\nOur books are available at our direct Print Sales Department and through selected representatives throughout the world.
\n\nBooks International
\n\nRepresentative for: Brunei, Cambodia, Indonesia, Indonesia, Laos, Malaysia, Myanmar, Philippines, Singapore, Thailand, Vietnam (ASEAN)
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On September, 29th 2006 he has won a post PhD fellowship from the university of Bologna (from October 2006 to October 2008), at the competitive examination he was ranked first in the industrial engineering area. He extensively served as referee for several international journals. He is author/coauthor of more than 100 research papers. He has been involved in some projects supported by MURST and European Community. 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He is a member of the Turkish Biochemical Society, American Chemical Society, and German Genetics society. Dr. Ekinci published around ninety scientific papers, reviews and book chapters, and presented several conferences to scientists. He has received numerous publication awards from several scientific councils. 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Radiotherapy and Nuclear Medicine Technology has always been my aspiration and my life. As years passed I accumulated a tremendous amount of skills and knowledge in Radiotherapy and Nuclear Medicine, Conventional Radiology, Radiation Protection, Bioinformatics Technology, PACS, Image processing, clinically and lecturing that will enable me to provide a valuable service to the community as a Researcher and Consultant in this field. My method of translating this into day to day in clinical practice is non-exhaustible and my habit of exchanging knowledge and expertise with others in those fields is the code and secret of success.",institutionString:null,institution:{name:"Majmaah University",country:{name:"Saudi Arabia"}}},{id:"313277",title:"Dr.",name:"Bartłomiej",middleName:null,surname:"Płaczek",slug:"bartlomiej-placzek",fullName:"Bartłomiej Płaczek",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/313277/images/system/313277.jpg",biography:"Bartłomiej Płaczek, MSc (2002), Ph.D. (2005), Habilitation (2016), is a professor at the University of Silesia, Institute of Computer Science, Poland, and an expert from the National Centre for Research and Development. His research interests include sensor networks, smart sensors, intelligent systems, and image processing with applications in healthcare and medicine. He is the author or co-author of more than seventy papers in peer-reviewed journals and conferences as well as the co-author of several books. He serves as a reviewer for many scientific journals, international conferences, and research foundations. Since 2010, Dr. Placzek has been a reviewer of grants and projects (including EU projects) in the field of information technologies.",institutionString:"University of Silesia",institution:{name:"University of Silesia",country:{name:"Poland"}}},{id:"35000",title:"Prof.",name:"Ulrich H.P",middleName:"H.P.",surname:"Fischer",slug:"ulrich-h.p-fischer",fullName:"Ulrich H.P Fischer",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/35000/images/3052_n.jpg",biography:"Academic and Professional Background\nUlrich H. P. has Diploma and PhD degrees in Physics from the Free University Berlin, Germany. He has been working on research positions in the Heinrich-Hertz-Institute in Germany. Several international research projects has been performed with European partners from France, Netherlands, Norway and the UK. He is currently Professor of Communications Systems at the Harz University of Applied Sciences, Germany.\n\nPublications and Publishing\nHe has edited one book, a special interest book about ‘Optoelectronic Packaging’ (VDE, Berlin, Germany), and has published over 100 papers and is owner of several international patents for WDM over POF key elements.\n\nKey Research and Consulting Interests\nUlrich’s research activity has always been related to Spectroscopy and Optical Communications Technology. Specific current interests include the validation of complex instruments, and the application of VR technology to the development and testing of measurement systems. He has been reviewer for several publications of the Optical Society of America\\'s including Photonics Technology Letters and Applied Optics.\n\nPersonal Interests\nThese include motor cycling in a very relaxed manner and performing martial arts.",institutionString:null,institution:{name:"Charité",country:{name:"Germany"}}},{id:"341622",title:"Ph.D.",name:"Eduardo",middleName:null,surname:"Rojas Alvarez",slug:"eduardo-rojas-alvarez",fullName:"Eduardo Rojas Alvarez",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/341622/images/15892_n.jpg",biography:null,institutionString:null,institution:{name:"University of Cuenca",country:{name:"Ecuador"}}},{id:"215610",title:"Prof.",name:"Muhammad",middleName:null,surname:"Sarfraz",slug:"muhammad-sarfraz",fullName:"Muhammad Sarfraz",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/215610/images/system/215610.jpeg",biography:"Muhammad Sarfraz is a professor in the Department of Information Science, Kuwait University. His research interests include computer graphics, computer vision, image processing, machine learning, pattern recognition, soft computing, data science, intelligent systems, information technology, and information systems. Prof. Sarfraz has been a keynote/invited speaker on various platforms around the globe. He has advised various students for their MSc and Ph.D. theses. He has published more than 400 publications as books, journal articles, and conference papers. He is a member of various professional societies and a chair and member of the International Advisory Committees and Organizing Committees of various international conferences. Prof. Sarfraz is also an editor-in-chief and editor of various international journals.",institutionString:"Kuwait University",institution:{name:"Kuwait University",country:{name:"Kuwait"}}},{id:"32650",title:"Prof.",name:"Lukas",middleName:"Willem",surname:"Snyman",slug:"lukas-snyman",fullName:"Lukas Snyman",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/32650/images/4136_n.jpg",biography:"Lukas Willem Snyman received his basic education at primary and high schools in South Africa, Eastern Cape. He enrolled at today's Nelson Metropolitan University and graduated from this university with a BSc in Physics and Mathematics, B.Sc Honors in Physics, MSc in Semiconductor Physics, and a Ph.D. in Semiconductor Physics in 1987. After his studies, he chose an academic career and devoted his energy to the teaching of physics to first, second, and third-year students. After positions as a lecturer at the University of Port Elizabeth, he accepted a position as Associate Professor at the University of Pretoria, South Africa.\r\n\r\nIn 1992, he motivates the concept of 'television and computer-based education” as means to reach large student numbers with only the best of teaching expertise and publishes an article on the concept in the SA Journal of Higher Education of 1993 (and later in 2003). The University of Pretoria subsequently approved a series of test projects on the concept with outreach to Mamelodi and Eerste Rust in 1993. In 1994, the University established a 'Unit for Telematic Education ' as a support section for multiple faculties at the University of Pretoria. In subsequent years, the concept of 'telematic education” subsequently becomes well established in academic circles in South Africa, grew in popularity, and is adopted by many universities and colleges throughout South Africa as a medium of enhancing education and training, as a method to reaching out to far out communities, and as a means to enhance study from the home environment.\r\n\r\nProfessor Snyman in subsequent years pursued research in semiconductor physics, semiconductor devices, microelectronics, and optoelectronics.\r\n\r\nIn 2000 he joined the TUT as a full professor. Here served for a period as head of the Department of Electronic Engineering. Here he makes contributions to solar energy development, microwave and optoelectronic device development, silicon photonics, as well as contributions to new mobile telecommunication systems and network planning in SA.\r\n\r\nCurrently, he teaches electronics and telecommunications at the TUT to audiences ranging from first-year students to Ph.D. level.\r\n\r\nFor his research in the field of 'Silicon Photonics” since 1990, he has published (as author and co-author) about thirty internationally reviewed articles in scientific journals, contributed to more than forty international conferences, about 25 South African provisional patents (as inventor and co-inventor), 8 PCT international patent applications until now. Of these, two USA patents applications, two European Patents, two Korean patents, and ten SA patents have been granted. A further 4 USA patents, 5 European patents, 3 Korean patents, 3 Chinese patents, and 3 Japanese patents are currently under consideration.\r\n\r\nRecently he has also published an extensive scholarly chapter in an internet open access book on 'Integrating Microphotonic Systems and MOEMS into standard Silicon CMOS Integrated circuitry”.\r\n\r\nFurthermore, Professor Snyman recently steered a new initiative at the TUT by introducing a 'Laboratory for Innovative Electronic Systems ' at the Department of Electrical Engineering. The model of this laboratory or center is to primarily combine outputs as achieved by high-level research with lower-level system development and entrepreneurship in a technical university environment. Students are allocated to projects at different levels with PhDs and Master students allocated to the generation of new knowledge and new technologies, while students at the diploma and Baccalaureus level are allocated to electronic systems development with a direct and a near application for application in industry or the commercial and public sectors in South Africa.\r\n\r\nProfessor Snyman received the WIRSAM Award of 1983 and the WIRSAM Award in 1985 in South Africa for best research papers by a young scientist at two international conferences on electron microscopy in South Africa. He subsequently received the SA Microelectronics Award for the best dissertation emanating from studies executed at a South African university in the field of Physics and Microelectronics in South Africa in 1987. In October of 2011, Professor Snyman received the prestigious Institutional Award for 'Innovator of the Year” for 2010 at the Tshwane University of Technology, South Africa. This award was based on the number of patents recognized and granted by local and international institutions as well as for his contributions concerning innovation at the TUT.",institutionString:null,institution:{name:"University of South Africa",country:{name:"South Africa"}}},{id:"317279",title:"Mr.",name:"Ali",middleName:"Usama",surname:"Syed",slug:"ali-syed",fullName:"Ali Syed",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/317279/images/16024_n.png",biography:"A creative, talented, and innovative young professional who is dedicated, well organized, and capable research fellow with two years of experience in graduate-level research, published in engineering journals and book, with related expertise in Bio-robotics, equally passionate about the aesthetics of the mechanical and electronic system, obtained expertise in the use of MS Office, MATLAB, SolidWorks, LabVIEW, Proteus, Fusion 360, having a grasp on python, C++ and assembly language, possess proven ability in acquiring research grants, previous appointments with social and educational societies with experience in administration, current affiliations with IEEE and Web of Science, a confident presenter at conferences and teacher in classrooms, able to explain complex information to audiences of all levels.",institutionString:null,institution:{name:"Air University",country:{name:"Pakistan"}}},{id:"75526",title:"Ph.D.",name:"Zihni Onur",middleName:null,surname:"Uygun",slug:"zihni-onur-uygun",fullName:"Zihni Onur Uygun",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/75526/images/12_n.jpg",biography:"My undergraduate education and my Master of Science educations at Ege University and at Çanakkale Onsekiz Mart University have given me a firm foundation in Biochemistry, Analytical Chemistry, Biosensors, Bioelectronics, Physical Chemistry and Medicine. After obtaining my degree as a MSc in analytical chemistry, I started working as a research assistant in Ege University Medical Faculty in 2014. In parallel, I enrolled to the MSc program at the Department of Medical Biochemistry at Ege University to gain deeper knowledge on medical and biochemical sciences as well as clinical chemistry in 2014. In my PhD I deeply researched on biosensors and bioelectronics and finished in 2020. Now I have eleven SCI-Expanded Index published papers, 6 international book chapters, referee assignments for different SCIE journals, one international patent pending, several international awards, projects and bursaries. In parallel to my research assistant position at Ege University Medical Faculty, Department of Medical Biochemistry, in April 2016, I also founded a Start-Up Company (Denosens Biotechnology LTD) by the support of The Scientific and Technological Research Council of Turkey. Currently, I am also working as a CEO in Denosens Biotechnology. The main purposes of the company, which carries out R&D as a research center, are to develop new generation biosensors and sensors for both point-of-care diagnostics; such as glucose, lactate, cholesterol and cancer biomarker detections. My specific experimental and instrumental skills are Biochemistry, Biosensor, Analytical Chemistry, Electrochemistry, Mobile phone based point-of-care diagnostic device, POCTs and Patient interface designs, HPLC, Tandem Mass Spectrometry, Spectrophotometry, ELISA.",institutionString:null,institution:{name:"Ege University",country:{name:"Turkey"}}},{id:"267434",title:"Dr.",name:"Rohit",middleName:null,surname:"Raja",slug:"rohit-raja",fullName:"Rohit Raja",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/267434/images/system/267434.jpg",biography:"Dr. Rohit Raja received Ph.D. in Computer Science and Engineering from Dr. CVRAMAN University in 2016. His main research interest includes Face recognition and Identification, Digital Image Processing, Signal Processing, and Networking. Presently he is working as Associate Professor in IT Department, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur (CG), India. He has authored several Journal and Conference Papers. He has good Academics & Research experience in various areas of CSE and IT. He has filed and successfully published 27 Patents. He has received many time invitations to be a Guest at IEEE Conferences. He has published 100 research papers in various International/National Journals (including IEEE, Springer, etc.) and Proceedings of the reputed International/ National Conferences (including Springer and IEEE). He has been nominated to the board of editors/reviewers of many peer-reviewed and refereed Journals (including IEEE, Springer).",institutionString:"Guru Ghasidas Vishwavidyalaya",institution:{name:"Guru Ghasidas Vishwavidyalaya",country:{name:"India"}}},{id:"246502",title:"Dr.",name:"Jaya T.",middleName:"T",surname:"Varkey",slug:"jaya-t.-varkey",fullName:"Jaya T. Varkey",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/246502/images/11160_n.jpg",biography:"Jaya T. Varkey, PhD, graduated with a degree in Chemistry from Cochin University of Science and Technology, Kerala, India. She obtained a PhD in Chemistry from the School of Chemical Sciences, Mahatma Gandhi University, Kerala, India, and completed a post-doctoral fellowship at the University of Minnesota, USA. She is a research guide at Mahatma Gandhi University and Associate Professor in Chemistry, St. Teresa’s College, Kochi, Kerala, India.\nDr. Varkey received a National Young Scientist award from the Indian Science Congress (1995), a UGC Research award (2016–2018), an Indian National Science Academy (INSA) Visiting Scientist award (2018–2019), and a Best Innovative Faculty award from the All India Association for Christian Higher Education (AIACHE) (2019). She Hashas received the Sr. Mary Cecil prize for best research paper three times. She was also awarded a start-up to develop a tea bag water filter. \nDr. Varkey has published two international books and twenty-seven international journal publications. She is an editorial board member for five international journals.",institutionString:"St. Teresa’s College",institution:null},{id:"250668",title:"Dr.",name:"Ali",middleName:null,surname:"Nabipour Chakoli",slug:"ali-nabipour-chakoli",fullName:"Ali Nabipour Chakoli",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/250668/images/system/250668.jpg",biography:"Academic Qualification:\r\n•\tPhD in Materials Physics and Chemistry, From: Sep. 2006, to: Sep. 2010, School of Materials Science and Engineering, Harbin Institute of Technology, Thesis: Structure and Shape Memory Effect of Functionalized MWCNTs/poly (L-lactide-co-ε-caprolactone) Nanocomposites. Supervisor: Prof. Wei Cai,\r\n•\tM.Sc in Applied Physics, From: 1996, to: 1998, Faculty of Physics & Nuclear Science, Amirkabir Uni. of Technology, Tehran, Iran, Thesis: Determination of Boron in Micro alloy Steels with solid state nuclear track detectors by neutron induced auto radiography, Supervisors: Dr. M. Hosseini Ashrafi and Dr. A. Hosseini.\r\n•\tB.Sc. in Applied Physics, From: 1991, to: 1996, Faculty of Physics & Nuclear Science, Amirkabir Uni. of Technology, Tehran, Iran, Thesis: Design of shielding for Am-Be neutron sources for In Vivo neutron activation analysis, Supervisor: Dr. M. Hosseini Ashrafi.\r\n\r\nResearch Experiences:\r\n1.\tNanomaterials, Carbon Nanotubes, Graphene: Synthesis, Functionalization and Characterization,\r\n2.\tMWCNTs/Polymer Composites: Fabrication and Characterization, \r\n3.\tShape Memory Polymers, Biodegradable Polymers, ORC, Collagen,\r\n4.\tMaterials Analysis and Characterizations: TEM, SEM, XPS, FT-IR, Raman, DSC, DMA, TGA, XRD, GPC, Fluoroscopy, \r\n5.\tInteraction of Radiation with Mater, Nuclear Safety and Security, NDT(RT),\r\n6.\tRadiation Detectors, Calibration (SSDL),\r\n7.\tCompleted IAEA e-learning Courses:\r\nNuclear Security (15 Modules),\r\nNuclear Safety:\r\nTSA 2: Regulatory Protection in Occupational Exposure,\r\nTips & Tricks: Radiation Protection in Radiography,\r\nSafety and Quality in Radiotherapy,\r\nCourse on Sealed Radioactive Sources,\r\nCourse on Fundamentals of Environmental Remediation,\r\nCourse on Planning for Environmental Remediation,\r\nKnowledge Management Orientation Course,\r\nFood Irradiation - Technology, Applications and Good Practices,\r\nEmployment:\r\nFrom 2010 to now: Academic staff, Nuclear Science and Technology Research Institute, Kargar Shomali, Tehran, Iran, P.O. Box: 14395-836.\r\nFrom 1997 to 2006: Expert of Materials Analysis and Characterization. Research Center of Agriculture and Medicine. Rajaeeshahr, Karaj, Iran, P. O. Box: 31585-498.",institutionString:"Atomic Energy Organization of Iran",institution:{name:"Atomic Energy Organization of Iran",country:{name:"Iran"}}},{id:"248279",title:"Dr.",name:"Monika",middleName:"Elzbieta",surname:"Machoy",slug:"monika-machoy",fullName:"Monika Machoy",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/248279/images/system/248279.jpeg",biography:"Monika Elżbieta Machoy, MD, graduated with distinction from the Faculty of Medicine and Dentistry at the Pomeranian Medical University in 2009, defended her PhD thesis with summa cum laude in 2016 and is currently employed as a researcher at the Department of Orthodontics of the Pomeranian Medical University. She expanded her professional knowledge during a one-year scholarship program at the Ernst Moritz Arndt University in Greifswald, Germany and during a three-year internship at the Technical University in Dresden, Germany. She has been a speaker at numerous orthodontic conferences, among others, American Association of Orthodontics, European Orthodontic Symposium and numerous conferences of the Polish Orthodontic Society. She conducts research focusing on the effect of orthodontic treatment on dental and periodontal tissues and the causes of pain in orthodontic patients.",institutionString:"Pomeranian Medical University",institution:{name:"Pomeranian Medical University",country:{name:"Poland"}}},{id:"252743",title:"Prof.",name:"Aswini",middleName:"Kumar",surname:"Kar",slug:"aswini-kar",fullName:"Aswini Kar",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/252743/images/10381_n.jpg",biography:"uploaded in cv",institutionString:null,institution:{name:"KIIT University",country:{name:"India"}}},{id:"204256",title:"Dr.",name:"Anil",middleName:"Kumar",surname:"Kumar Sahu",slug:"anil-kumar-sahu",fullName:"Anil Kumar Sahu",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/204256/images/14201_n.jpg",biography:"I have nearly 11 years of research and teaching experience. I have done my master degree from University Institute of Pharmacy, Pt. Ravi Shankar Shukla University, Raipur, Chhattisgarh India. I have published 16 review and research articles in international and national journals and published 4 chapters in IntechOpen, the world’s leading publisher of Open access books. I have presented many papers at national and international conferences. I have received research award from Indian Drug Manufacturers Association in year 2015. My research interest extends from novel lymphatic drug delivery systems, oral delivery system for herbal bioactive to formulation optimization.",institutionString:null,institution:{name:"Chhattisgarh Swami Vivekanand Technical University",country:{name:"India"}}},{id:"253468",title:"Dr.",name:"Mariusz",middleName:null,surname:"Marzec",slug:"mariusz-marzec",fullName:"Mariusz Marzec",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/253468/images/system/253468.png",biography:"An assistant professor at Department of Biomedical Computer Systems, at Institute of Computer Science, Silesian University in Katowice. Scientific interests: computer analysis and processing of images, biomedical images, databases and programming languages. He is an author and co-author of scientific publications covering analysis and processing of biomedical images and development of database systems.",institutionString:"University of Silesia",institution:{name:"University of Silesia",country:{name:"Poland"}}},{id:"212432",title:"Prof.",name:"Hadi",middleName:null,surname:"Mohammadi",slug:"hadi-mohammadi",fullName:"Hadi Mohammadi",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/212432/images/system/212432.jpeg",biography:"Dr. Hadi Mohammadi is a biomedical engineer with hands-on experience in the design and development of many engineering structures and medical devices through various projects that he has been involved in over the past twenty years. Dr. Mohammadi received his BSc. and MSc. degrees in Mechanical Engineering from Sharif University of Technology, Tehran, Iran, and his PhD. degree in Biomedical Engineering (biomaterials) from the University of Western Ontario. He was a postdoctoral trainee for almost four years at University of Calgary and Harvard Medical School. He is an industry innovator having created the technology to produce lifelike synthetic platforms that can be used for the simulation of almost all cardiovascular reconstructive surgeries. He’s been heavily involved in the design and development of cardiovascular devices and technology for the past 10 years. He is currently an Assistant Professor with the University of British Colombia, Canada.",institutionString:"University of British Columbia",institution:{name:"University of British Columbia",country:{name:"Canada"}}},{id:"254463",title:"Prof.",name:"Haisheng",middleName:null,surname:"Yang",slug:"haisheng-yang",fullName:"Haisheng Yang",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/254463/images/system/254463.jpeg",biography:"Haisheng Yang, Ph.D., Professor and Director of the Department of Biomedical Engineering, College of Life Science and Bioengineering, Beijing University of Technology. He received his Ph.D. degree in Mechanics/Biomechanics from Harbin Institute of Technology (jointly with University of California, Berkeley). Afterwards, he worked as a Postdoctoral Research Associate in the Purdue Musculoskeletal Biology and Mechanics Lab at the Department of Basic Medical Sciences, Purdue University, USA. He also conducted research in the Research Centre of Shriners Hospitals for Children-Canada at McGill University, Canada. Dr. Yang has over 10 years research experience in orthopaedic biomechanics and mechanobiology of bone adaptation and regeneration. He earned an award from Beijing Overseas Talents Aggregation program in 2017 and serves as Beijing Distinguished Professor.",institutionString:null,institution:{name:"Beijing University of Technology",country:{name:"China"}}},{id:"89721",title:"Dr.",name:"Mehmet",middleName:"Cuneyt",surname:"Ozmen",slug:"mehmet-ozmen",fullName:"Mehmet Ozmen",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/89721/images/7289_n.jpg",biography:null,institutionString:null,institution:{name:"Gazi University",country:{name:"Turkey"}}},{id:"265335",title:"Mr.",name:"Stefan",middleName:"Radnev",surname:"Stefanov",slug:"stefan-stefanov",fullName:"Stefan Stefanov",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/265335/images/7562_n.jpg",biography:null,institutionString:null,institution:{name:"Medical University Plovdiv",country:{name:"Bulgaria"}}},{id:"242893",title:"Ph.D. Student",name:"Joaquim",middleName:null,surname:"De Moura",slug:"joaquim-de-moura",fullName:"Joaquim De Moura",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/242893/images/7133_n.jpg",biography:"Joaquim de Moura received his degree in Computer Engineering in 2014 from the University of A Coruña (Spain). In 2016, he received his M.Sc degree in Computer Engineering from the same university. He is currently pursuing his Ph.D degree in Computer Science in a collaborative project between ophthalmology centers in Galicia and the University of A Coruña. His research interests include computer vision, machine learning algorithms and analysis and medical imaging processing of various kinds.",institutionString:null,institution:{name:"University of A Coruña",country:{name:"Spain"}}},{id:"294334",title:"B.Sc.",name:"Marc",middleName:null,surname:"Bruggeman",slug:"marc-bruggeman",fullName:"Marc Bruggeman",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/294334/images/8242_n.jpg",biography:"Chemical engineer graduate, with a passion for material science and specific interest in polymers - their near infinite applications intrigue me. \n\nI plan to continue my scientific career in the field of polymeric biomaterials as I am fascinated by intelligent, bioactive and biomimetic materials for use in both consumer and medical applications.",institutionString:null,institution:null},{id:"255757",title:"Dr.",name:"Igor",middleName:"Victorovich",surname:"Lakhno",slug:"igor-lakhno",fullName:"Igor Lakhno",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/255757/images/system/255757.jpg",biography:"Igor Victorovich Lakhno was born in 1971 in Kharkiv (Ukraine). \nMD – 1994, Kharkiv National Medical Univesity.\nOb&Gyn; – 1997, master courses in Kharkiv Medical Academy of Postgraduate Education.\nPh.D. – 1999, Kharkiv National Medical Univesity.\nDSC – 2019, PL Shupik National Academy of Postgraduate Education \nProfessor – 2021, Department of Obstetrics and Gynecology of VN Karazin Kharkiv National University\nHead of Department – 2021, Department of Perinatology, Obstetrics and gynecology of Kharkiv Medical Academy of Postgraduate Education\nIgor Lakhno has been graduated from international training courses on reproductive medicine and family planning held at Debrecen University (Hungary) in 1997. Since 1998 Lakhno Igor has worked as an associate professor in the department of obstetrics and gynecology of VN Karazin National University and an associate professor of the perinatology, obstetrics, and gynecology department of Kharkiv Medical Academy of Postgraduate Education. Since June 2019 he’s been a professor in the department of obstetrics and gynecology of VN Karazin National University and a professor of the perinatology, obstetrics, and gynecology department. He’s affiliated with Kharkiv Medical Academy of Postgraduate Education as a Head of Department from November 2021. Igor Lakhno has participated in several international projects on fetal non-invasive electrocardiography (with Dr. J. A. Behar (Technion), Prof. D. Hoyer (Jena University), and José Alejandro Díaz Méndez (National Institute of Astrophysics, Optics, and Electronics, Mexico). He’s an author of about 200 printed works and there are 31 of them in Scopus or Web of Science databases. Igor Lakhno is a member of the Editorial Board of Reproductive Health of Woman, Emergency Medicine, and Technology Transfer Innovative Solutions in Medicine (Estonia). He is a medical Editor of “Z turbotoyu pro zhinku”. Igor Lakhno is a reviewer of the Journal of Obstetrics and Gynaecology (Taylor and Francis), British Journal of Obstetrics and Gynecology (Wiley), Informatics in Medicine Unlocked (Elsevier), The Journal of Obstetrics and Gynecology Research (Wiley), Endocrine, Metabolic & Immune Disorders-Drug Targets (Bentham Open), The Open Biomedical Engineering Journal (Bentham Open), etc. He’s defended a dissertation for a DSc degree “Pre-eclampsia: prediction, prevention, and treatment”. Three years ago Igor Lakhno has participated in a training course on innovative technologies in medical education at Lublin Medical University (Poland). Lakhno Igor has participated as a speaker in several international conferences and congresses (International Conference on Biological Oscillations April 10th-14th 2016, Lancaster, UK, The 9th conference of the European Study Group on Cardiovascular Oscillations). His main scientific interests: are obstetrics, women’s health, fetal medicine, and cardiovascular medicine. \nIgor Lakhno is a consultant at Kharkiv municipal perinatal center. He’s graduated from training courses on endoscopy in gynecology. He has 28 years of practical experience in the field.",institutionString:null,institution:null},{id:"244950",title:"Dr.",name:"Salvatore",middleName:null,surname:"Di Lauro",slug:"salvatore-di-lauro",fullName:"Salvatore Di Lauro",position:null,profilePictureURL:"https://intech-files.s3.amazonaws.com/0030O00002bSF1HQAW/ProfilePicture%202021-12-20%2014%3A54%3A14.482",biography:"Name:\n\tSALVATORE DI LAURO\nAddress:\n\tHospital Clínico Universitario Valladolid\nAvda Ramón y Cajal 3\n47005, Valladolid\nSpain\nPhone number: \nFax\nE-mail:\n\t+34 983420000 ext 292\n+34 983420084\nsadilauro@live.it\nDate and place of Birth:\nID Number\nMedical Licence \nLanguages\t09-05-1985. Villaricca (Italy)\n\nY1281863H\n474707061\nItalian (native language)\nSpanish (read, written, spoken)\nEnglish (read, written, spoken)\nPortuguese (read, spoken)\nFrench (read)\n\t\t\nCurrent position (title and company)\tDate (Year)\nVitreo-Retinal consultant in ophthalmology. Hospital Clinico Universitario Valladolid. Sacyl. National Health System.\nVitreo-Retinal consultant in ophthalmology. Instituto Oftalmologico Recoletas. Red Hospitalaria Recoletas. Private practise.\t2017-today\n\n2019-today\n\t\n\t\nEducation (High school, university and postgraduate training > 3 months)\tDate (Year)\nDegree in Medicine and Surgery. University of Neaples 'Federico II”\nResident in Opthalmology. Hospital Clinico Universitario Valladolid\nMaster in Vitreo-Retina. IOBA. University of Valladolid\nFellow of the European Board of Ophthalmology. Paris\nMaster in Research in Ophthalmology. University of Valladolid\t2003-2009\n2012-2016\n2016-2017\n2016\n2012-2013\n\t\nEmployments (company and positions)\tDate (Year)\nResident in Ophthalmology. Hospital Clinico Universitario Valladolid. Sacyl.\nFellow in Vitreo-Retina. IOBA. University of Valladolid\nVitreo-Retinal consultant in ophthalmology. Hospital Clinico Universitario Valladolid. Sacyl. National Health System.\nVitreo-Retinal consultant in ophthalmology. Instituto Oftalmologico Recoletas. Red Hospitalaria Recoletas. \n\t2012-2016\n2016-2017\n2017-today\n\n2019-Today\n\n\n\t\nClinical Research Experience (tasks and role)\tDate (Year)\nAssociated investigator\n\n' FIS PI20/00740: DESARROLLO DE UNA CALCULADORA DE RIESGO DE\nAPARICION DE RETINOPATIA DIABETICA BASADA EN TECNICAS DE IMAGEN MULTIMODAL EN PACIENTES DIABETICOS TIPO 1. Grant by: Ministerio de Ciencia e Innovacion \n\n' (BIO/VA23/14) Estudio clínico multicéntrico y prospectivo para validar dos\nbiomarcadores ubicados en los genes p53 y MDM2 en la predicción de los resultados funcionales de la cirugía del desprendimiento de retina regmatógeno. Grant by: Gerencia Regional de Salud de la Junta de Castilla y León.\n' Estudio multicéntrico, aleatorizado, con enmascaramiento doble, en 2 grupos\nparalelos y de 52 semanas de duración para comparar la eficacia, seguridad e inmunogenicidad de SOK583A1 respecto a Eylea® en pacientes con degeneración macular neovascular asociada a la edad' (CSOK583A12301; N.EUDRA: 2019-004838-41; FASE III). Grant by Hexal AG\n\n' Estudio de fase III, aleatorizado, doble ciego, con grupos paralelos, multicéntrico para comparar la eficacia y la seguridad de QL1205 frente a Lucentis® en pacientes con degeneración macular neovascular asociada a la edad. (EUDRACT: 2018-004486-13). Grant by Qilu Pharmaceutical Co\n\n' Estudio NEUTON: Ensayo clinico en fase IV para evaluar la eficacia de aflibercept en pacientes Naive con Edema MacUlar secundario a Oclusion de Vena CenTral de la Retina (OVCR) en regimen de tratamientO iNdividualizado Treat and Extend (TAE)”, (2014-000975-21). Grant by Fundacion Retinaplus\n\n' Evaluación de la seguridad y bioactividad de anillos de tensión capsular en conejo. Proyecto Procusens. Grant by AJL, S.A.\n\n'Estudio epidemiológico, prospectivo, multicéntrico y abierto\\npara valorar la frecuencia de la conjuntivitis adenovírica diagnosticada mediante el test AdenoPlus®\\nTest en pacientes enfermos de conjuntivitis aguda”\\n. National, multicenter study. Grant by: NICOX.\n\nEuropean multicentric trial: 'Evaluation of clinical outcomes following the use of Systane Hydration in patients with dry eye”. Study Phase 4. Grant by: Alcon Labs'\n\nVLPs Injection and Activation in a Rabbit Model of Uveal Melanoma. Grant by Aura Bioscience\n\nUpdating and characterization of a rabbit model of uveal melanoma. Grant by Aura Bioscience\n\nEnsayo clínico en fase IV para evaluar las variantes genéticas de la vía del VEGF como biomarcadores de eficacia del tratamiento con aflibercept en pacientes con degeneración macular asociada a la edad (DMAE) neovascular. Estudio BIOIMAGE. IMO-AFLI-2013-01\n\nEstudio In-Eye:Ensayo clínico en fase IV, abierto, aleatorizado, de 2 brazos,\nmulticçentrico y de 12 meses de duración, para evaluar la eficacia y seguridad de un régimen de PRN flexible individualizado de 'esperar y extender' versus un régimen PRN según criterios de estabilización mediante evaluaciones mensuales de inyecciones intravítreas de ranibizumab 0,5 mg en pacientes naive con neovascularización coriodea secunaria a la degeneración macular relacionada con la edad. CP: CRFB002AES03T\n\nTREND: Estudio Fase IIIb multicéntrico, randomizado, de 12 meses de\nseguimiento con evaluador de la agudeza visual enmascarado, para evaluar la eficacia y la seguridad de ranibizumab 0.5mg en un régimen de tratar y extender comparado con un régimen mensual, en pacientes con degeneración macular neovascular asociada a la edad. CP: CRFB002A2411 Código Eudra CT:\n2013-002626-23\n\n\n\nPublications\t\n\n2021\n\n\n\n\n2015\n\n\n\n\n2021\n\n\n\n\n\n2021\n\n\n\n\n2015\n\n\n\n\n2015\n\n\n2014\n\n\n\n\n2015-16\n\n\n\n2015\n\n\n2014\n\n\n2014\n\n\n\n\n2014\n\n\n\n\n\n\n\n2014\n\nJose Carlos Pastor; Jimena Rojas; Salvador Pastor-Idoate; Salvatore Di Lauro; Lucia Gonzalez-Buendia; Santiago Delgado-Tirado. Proliferative vitreoretinopathy: A new concept of disease pathogenesis and practical\nconsequences. Progress in Retinal and Eye Research. 51, pp. 125 - 155. 03/2016. DOI: 10.1016/j.preteyeres.2015.07.005\n\n\nLabrador-Velandia S; Alonso-Alonso ML; Di Lauro S; García-Gutierrez MT; Srivastava GK; Pastor JC; Fernandez-Bueno I. Mesenchymal stem cells provide paracrine neuroprotective resources that delay degeneration of co-cultured organotypic neuroretinal cultures.Experimental Eye Research. 185, 17/05/2019. DOI: 10.1016/j.exer.2019.05.011\n\nSalvatore Di Lauro; Maria Teresa Garcia Gutierrez; Ivan Fernandez Bueno. Quantification of pigment epithelium-derived factor (PEDF) in an ex vivo coculture of retinal pigment epithelium cells and neuroretina.\nJournal of Allbiosolution. 2019. ISSN 2605-3535\n\nSonia Labrador Velandia; Salvatore Di Lauro; Alonso-Alonso ML; Tabera Bartolomé S; Srivastava GK; Pastor JC; Fernandez-Bueno I. Biocompatibility of intravitreal injection of human mesenchymal stem cells in immunocompetent rabbits. Graefe's archive for clinical and experimental ophthalmology. 256 - 1, pp. 125 - 134. 01/2018. DOI: 10.1007/s00417-017-3842-3\n\n\nSalvatore Di Lauro, David Rodriguez-Crespo, Manuel J Gayoso, Maria T Garcia-Gutierrez, J Carlos Pastor, Girish K Srivastava, Ivan Fernandez-Bueno. A novel coculture model of porcine central neuroretina explants and retinal pigment epithelium cells. Molecular Vision. 2016 - 22, pp. 243 - 253. 01/2016.\n\nSalvatore Di Lauro. Classifications for Proliferative Vitreoretinopathy ({PVR}): An Analysis of Their Use in Publications over the Last 15 Years. Journal of Ophthalmology. 2016, pp. 1 - 6. 01/2016. DOI: 10.1155/2016/7807596\n\nSalvatore Di Lauro; Rosa Maria Coco; Rosa Maria Sanabria; Enrique Rodriguez de la Rua; Jose Carlos Pastor. Loss of Visual Acuity after Successful Surgery for Macula-On Rhegmatogenous Retinal Detachment in a Prospective Multicentre Study. Journal of Ophthalmology. 2015:821864, 2015. DOI: 10.1155/2015/821864\n\nIvan Fernandez-Bueno; Salvatore Di Lauro; Ivan Alvarez; Jose Carlos Lopez; Maria Teresa Garcia-Gutierrez; Itziar Fernandez; Eva Larra; Jose Carlos Pastor. Safety and Biocompatibility of a New High-Density Polyethylene-Based\nSpherical Integrated Porous Orbital Implant: An Experimental Study in Rabbits. Journal of Ophthalmology. 2015:904096, 2015. DOI: 10.1155/2015/904096\n\nPastor JC; Pastor-Idoate S; Rodríguez-Hernandez I; Rojas J; Fernandez I; Gonzalez-Buendia L; Di Lauro S; Gonzalez-Sarmiento R. Genetics of PVR and RD. Ophthalmologica. 232 - Suppl 1, pp. 28 - 29. 2014\n\nRodriguez-Crespo D; Di Lauro S; Singh AK; Garcia-Gutierrez MT; Garrosa M; Pastor JC; Fernandez-Bueno I; Srivastava GK. Triple-layered mixed co-culture model of RPE cells with neuroretina for evaluating the neuroprotective effects of adipose-MSCs. Cell Tissue Res. 358 - 3, pp. 705 - 716. 2014.\nDOI: 10.1007/s00441-014-1987-5\n\nCarlo De Werra; Salvatore Condurro; Salvatore Tramontano; Mario Perone; Ivana Donzelli; Salvatore Di Lauro; Massimo Di Giuseppe; Rosa Di Micco; Annalisa Pascariello; Antonio Pastore; Giorgio Diamantis; Giuseppe Galloro. Hydatid disease of the liver: thirty years of surgical experience.Chirurgia italiana. 59 - 5, pp. 611 - 636.\n(Italia): 2007. ISSN 0009-4773\n\nChapters in books\n\t\n' Salvador Pastor Idoate; Salvatore Di Lauro; Jose Carlos Pastor Jimeno. PVR: Pathogenesis, Histopathology and Classification. Proliferative Vitreoretinopathy with Small Gauge Vitrectomy. Springer, 2018. ISBN 978-3-319-78445-8\nDOI: 10.1007/978-3-319-78446-5_2. \n\n' Salvatore Di Lauro; Maria Isabel Lopez Galvez. Quistes vítreos en una mujer joven. Problemas diagnósticos en patología retinocoroidea. Sociedad Española de Retina-Vitreo. 2018.\n\n' Salvatore Di Lauro; Salvador Pastor Idoate; Jose Carlos Pastor Jimeno. iOCT in PVR management. OCT Applications in Opthalmology. pp. 1 - 8. INTECH, 2018. DOI: 10.5772/intechopen.78774.\n\n' Rosa Coco Martin; Salvatore Di Lauro; Salvador Pastor Idoate; Jose Carlos Pastor. amponadores, manipuladores y tinciones en la cirugía del traumatismo ocular.Trauma Ocular. Ponencia de la SEO 2018..\n\n' LOPEZ GALVEZ; DI LAURO; CRESPO. OCT angiografia y complicaciones retinianas de la diabetes. PONENCIA SEO 2021, CAPITULO 20. (España): 2021.\n\n' Múltiples desprendimientos neurosensoriales bilaterales en paciente joven. Enfermedades Degenerativas De Retina Y Coroides. SERV 04/2016. \n' González-Buendía L; Di Lauro S; Pastor-Idoate S; Pastor Jimeno JC. Vitreorretinopatía proliferante (VRP) e inflamación: LA INFLAMACIÓN in «INMUNOMODULADORES Y ANTIINFLAMATORIOS: MÁS ALLÁ DE LOS CORTICOIDES. 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