{"paper_id":"b4435e46-2cfa-434e-be45-86c660801c8c","body_text":"1 \n \n \n \nMesenchymal stem cells: a promising tool for targeted gene therapy of Endometriosis \nAghila Rani Koippallil Gopalakrishnan1, Uday Kishore2 and Taruna Madan1* \n1Department of Innate Immunity, National Institute for Research in Reproductive Health \n(ICMR), Parel, Mumbai, India  \n2 Biosciences, College of Health and Life Science, Brunel University London, Uxbridge, \nUB83PH, United Kingdom.  \n \n \n* Corresponding author:   \nDr. Taruna Madan, Department of Innate Immunity,  \nNational Institute for Research in Reproductive Health (ICMR), \nJ. M. Street, Parel, Mumbai, India 400012.  \nTel: 00-91-22-24192049  \ne-mail: taruna_m@hotmail.com \n \n \n \n \n \n\n2 \n \nAbstract \nEndometriosis is a leading, benign gynaecological disorder around the world. Last few years \nhave witnessed tremendous growth in the field of endometriosis and endometrial stem cell \n(EnSC) research. Despite advancements in the biology and pathology of endometriosis, \ndisease recurrence is still an enigma. Gene therapy holds promise in treating many pathologic \nconditions including endometriosis. M esenchymal stem cells (MSCs) serve as ideal \ncandidates for regenerative medicine and cell based therapies.  Owing to their specificity to \nthe endometrium, residing endometrial MSC populations could be utilized as ideal candidates \nfor targeting endometrial disorders.  Recently, we demonstrated  their flexibili ty for gene \ntransduction using adenoviral vectors. The review highlight s the potential  of endometrial \nMSCs in devising targeted gene therapies for endometriosis.   \nKey words: Mesenchymal stem cells, gene therapy, endometriosis, angiogenesis, endometrial \nstem cells \n \n \n \n \n \n \n \n \n \n\n3 \n \nEndometriosis: a brief overview \nEndometriosis is a growing  gynaecological concern around the world with an alarming \nimpact on the reproductive function and social life of the affected women [1]. Symptoms of \nthe disease include  chronic pelvic pain, he avy menstrual bleeding, fatigue, dysmenorrhoea \nand dyspareunia [2]. The disease is also associated with female infertility with a prevalence \nrate of 30 to 50% world wide. Although most of the identified lesions are perpetually benign, \nreports suggest that in some rare instances, atypical lesions can lead to certain types of \novarian cancer s [3].  Long-term treatment for endometriosis includes pain management, \nregulation of estrogen levels, removal of ectopic endometrial tissue by laparoscopy/ \nlaparotomy, and hormonal therapies . Some of the  hormonal therapies are associated with \nserious hypoestrogenic side effects, such as bone mineral density loss , weight gain  and high \nrates of recurrence  [4, 5] . They are also ineffective in treating  endometriosis-associated \ninfertility and there is often recurrence of pelvic pain after the discontinuation of therapy [4, \n6]. Poor understanding of the disease pathogenesis poses technical limitations in designing \nappropriate treatment strategi es for endometriosis . The pathogenesis of endometriosis is \nattributed to a number of factors such as  retrograde menstruation, coelomic metaplasia, \nembryonic cell rest  and lymphatic/vascular dissemination of endometrial cells  [7, 8]. Other \npotential contributing factors include dysfunctional immune response, genetic predisposition, \nand aberrant peritoneal environment  that favour establishment and progression of \nendometriotic lesions  [7]. Recent evidences support  a possible involvement of endometrial \nstem/progenitor cells in the development of endometriosis [8-11].  \nRegenerating mesenchymal stem cell  (MSC) populations are identified both in the healthy \nendometrial (eutopic  MSCs) and endometriotic tissues (ectopic MSCs) [9, 10] . Eutopic \nMSCs reside in the endometrial lining of the uterus, whereas ectopic MSCs are isolated from \nthe growing endometriotic implants. Comparative studies have shown that ectopic MSCs are \n\n4 \n \nmore proliferative, migratory and angiogenic than eutopic MSCs obtained from the \nendometrial lining of same patient or control MSCs from healthy individuals without \nendometriosis [9, 10]. Hence, endometriosis has been described as an end result of an ectopic \nstem cell differentiation process. On the other hand, eutopic MSCs could be considered as the \nbest suited vehicle for treating endometrial disorders, as they are specific to the endometrium. \nIt is advantageous to use the same tissue stem cells as they would survive and function better \nthan MSCs from any other sources. In addition, it is possible that eutopic MSCs may take a \nlesser time -lag in adjusting to the endometrium microenvironment than MSCs of other \norigins, and therefore, enhancing its therapeutic efficacy.  \nMSCs have been used in a number of trials for corrective therapies using gene modification \ntechniques [12, 1 3]. MSC-mediated gene therapies possess certain unique advantages over \ndirect gene transfer s into the body ; these include  homing towards the site of injury, ease of \nhandling in culture conditions , weak immunogenicity and suitability for gene transduction \n[14]. A recent study from our group has indicated that MSC mediated gene therapy could be \napplied to endometriosis  [15]. The focus of this  review is to highlight the efficacy and \nbenefits of using residing eutopic MSC  populations as novel gene therapy tools for \nendometriosis.  \nEndometrial stem cells: a promising therapeutic tool \nThe last decade has  witnessed great progress in the endometrial stem cell research . Their \npresence in the human uterine tissue was first reported by Chan et al  in 2004  [16]. \nSubsequently, a number of studies reported the presence of resident MSC populations in both \nhuman and mouse uteri  [17 – 19].  An ever increasing number of FDA-approved clinical \ntrials that aim to treat a number of pathologies reflect the rise in demand for an ideal source \nof adult stem cells for therapeutic purposes. Endometrium , which undergoes nearly 400 \n\n5 \n \ncycles of compl ete regeneration and differentiation during each menstrual cycle , is thus an \nattractive and convenient source for regenerating adult stem cell populations [20].  \nHuman endometrium regenerates on a cyclic basis from underlying candidate endometrial \nstem/progenitors [21]. In vivo reports suggest the ability of endometrial stem/progenitor cells \nto develop human endometrium on subcutaneous injection in NOD -SCID mice [2 2, 23]. It \nhas been reported that the morphological and functional features of human  endometrium \ncould be reproduced in murine models using xenotransplantation techniques [ 24]. When \nhuman endometrial cells (epithelial, stromal, endothelial and immune cells) were \nxenotransplanted under the kidney capsule in severely immunodeficient mice, regeneration of \na functional endometrial tissue that mimics normal endometrium was observed. The \nengrafted human tissue formed chimeric functional blood vessels within the mouse \nendothelium validating the idea that human endothelial cells/progenitors derive d from the \nendometrium can migrate, invade and form vasculature in host tissues of different species \n[24]. Endometrial Stem Cells (EnSCs) are broadly classified into epithelial progenitor cells, \nMSCs, endothelial progenitor cells and endometrial side popul ation (SP) cells [21]. These \nstem cells are the likely precursors of endom etrial epithelial/stromal fibroblast cells [21]. The \nepithelial and stromal stem cell -like precursors have high proliferative potential and they \nundergo up to 30–32 population doublings before senescence, whereas endometrial non -stem \nepithelial and stromal cells undergo approximately 12 population doublings [9, 17]. Like their \ncounterparts, bone marrow stem cells (BMSCs)  [25], EnSCs could be  identified using \nspecific set of markers, such as CD9, CD13, CD14, CD29, CD31, CD44, CD73, CD90,  \nCD105, CD117, CD133 and CD146. They lack in expression for STRO-1, CD31 and CD34. \nAmong the different populations that are identified as endometrial stem/progenitor cells, \nendometrial side population (SP) cells [26] and MSCs [20] have drawn much attention  in \nrecent years .  A number of studies have suggested that EnSCs possess sid e population \n\n6 \n \nphenotype and are characterized by their ability to exclude the DNA -binding dye Hoechst \n33343 owing to presence of ATP -binding cassett e transporter proteins [22, 27, 28 ]. \nEndometrial SP cells are identified both in the epithelial and stromal compartment of the \nendometrium and endometriotic tissues [26, 28, 29]. Freshly isolated and in vitro cultures of \nEnSCs have been report ed to comprise up to 5% of SP cells [28, 30, 31]. Endometrial SP \ncells, both  from the stromal and epithelial compartments , displayed genotypic, phenotypic \nand functional features of somatic stem cells [22]. However, their therapeutic potential is yet \nto be explored further.  \nGargett et al identified the presence of regenerating MSC populations in the culture of freshly \nisolated endometrial stromal cells [17]. The study demonstrated that endometrial stromal cells \nexhibit MSC properties and co-express perivascular cell markers, such as CD146 and \nplatelet-derived growth factor -receptor β (PDGF -Rβ). CD146+ PDGFR+ EnSCs can \ndifferentiate into mesenchymal lineages, such as adipocytes, smooth muscle cells, \nchondrocytes and osteoblasts in vitro [17].  EnSCs can be grown extensively and maintained \nin culture for up  to 40 passages  [17]. Under specific conditions, these cells can differentiate \ninto various lineages such as chondrogenic, osteogenic, adipogenic, angiogenic and myogenic \n[9, 10, 1 7]. In addition to the above reports, a couple of studies  showed that bone marrow-\nderived stem cells (BMDSCs) contribute to the repair and regeneration of endometrial tissues \n[32, 33 ]. CD133(+) BMDSCs , when injected into a murine model of A sherman syndrome \n(AS), showed cell engraftment and proliferation around endometrial vessels [33]. The studies \nsuggest that the residing endometrial stem cell populations could be  BMDSCs that had \nmigrated under stimulating conditions and colonised in the endometrial tissue. \nThe therapeutic usefulness of EnSCs has been described by a number of  in vitro [34, 35] and \nin vivo studies [36, 37], and a few clinical trials  have also been reported [38 - 40]. In vivo \n\n7 \n \nstudies report ed their successful use in the treatment of Duchenne muscular dystrophy \n(DMD), cardiac, neural and bone regeneration, pancreatic differentiation, tissue engineering  \nand g lioma models [21]. Successful clinical trials include their use in mult iple sclerosis, \nischemic cardiomyopathy and DMD [21].  \nEndometrial Stem Cells: are they flexible tools for gene therapy? \nGene therapy holds an exciting promise for the treatment of numerous disorders ; a couple of \nanimal studies and in vitro experiments have indicated its application in endometriosis [5, 15, \n41, 42 ]. Potential targets for gene therapy of endometriosis include  genes related to \nangiogenesis, hormonal balance and inflammatory mediators  [43]. The advantages of using \ngene therapy for endometriosis include site-specific action of therapeutic agents and \navoidance of adverse side effects of conventional estrogen therapy and disease recurrence.  \nGene therapy trials for endometriosis are mentioned later in the article. Globally, nearly 600 \nclinical trials  have been registered that use MSCs for the treatment of a wide range of \nimmune and degenerative disorders  [44]. The clonogenic, immunogenic and differentiation \npotentials of MSCs offer the flexibility of th ese cells for use in such interventions [9, 1 8]. \nEndometrial (eutopic) MSCs serve as a powerful gene therapy tool due to their characteristic \nanti-inflammatory, immunosuppressive and tissue reparative properties [9].  Like MSCs from \nother sources, eutopic MSCs have also been extensively characterized for their immunogenic \nproperties [9, 10, 1 8].  We have earlier explored the immunosuppressive and \nimmunomodulatory features of eutopic and ectopic MSCs using peripheral blood \nmononuclear cells (P BMCs) [9].  In vitro co-culture experiments involving human eutopic \nMSCs and mitogen -activated PBMCs resulted in approximately 50% reduction in the \nproliferation of PBMCs , whereas the same was observed to be comprom ised in ectopic \nMSCs. We reported the immunosuppressive properties of eutopic MSCs and their feasibility \nin utilizing for cell transplantation studies [9]. Recently, we explored the therapeutic potential \n\n8 \n \nof eutopic MSCs in devising a targeted anti -angiogenic therapy for endometriosis  [15]. \nWomen with endometriosis exhibit enhanced angiogenesis at the ectopic lesion sites [10, 15]. \nHence, we focused on targeting angiogenesis in order to curb disease progression in \nendometriosis. Eutopic MSCs isolated from human endometrial biopsy specimen s were \ntransduced with a denoviral vectors expressing the anti -angiogenic factor, soluble truncated \nVEGF receptor-1, sFLT-1(Ad-sflt-1). In Ad-sflt-1 vector, sFlt-1 (soluble fms related tyrosine \nkinase 1) expression was under the control of cytomegalovirus (CMV) promoter. The vector \nwas propagated using human embryonic kidney 293 (HEK 293) cell lines and purified before \ntransducing to eutopic MSCs.  These experiments, for the first time, reported the flexibility of \neutopic MSCs to undergo any vector mediated transduction. The transduced cells were able \nto express MSC markers confirming their phenotypic stability following transduction \nprocedures.  The study demonstrated that eutopic MSCs could be genetically manipulated to \nexpress soluble Flt-1. Genetically manipulated eutopic MSCs expressed and secreted s Flt-1, \nand their therapeutic anti -angiogenic ability was validated in a SCID mouse model of \nendometriosis. Endometriosis was created subcutaneously in SCID mouse models and \ntherapeutic MSCs were administered intravenously. We were abl e to identify the presence of \nsFlt-1 secreting MSCs at target sites i n the treated group  [15]. Secreted sFlt-1 arrested lesion \ngrowth and angiogenesis and impaired expression of VEGF (vascular endothelial growth  \nfactor) and MMPs  (matrix metallo proteinases) . Th is study demonstrate d the success of a \nnovel strategy employing genetically manipulated endometrial MSCs for the treatment of \nendometriosis (Fig. 1).  \nAn effective anti -angiogenic agent should be able to inhibit angiogenesis both in the newly \ndeveloping/nascent lesions as well as  established lesions.  Usually, in experimental  \nendometriosis, anti -angiogenic reagents were administered in three different ways; (1) pre-\ntreatment of the study models before endometrial implantation ; (2) at the time of \n\n9 \n \ntransplantation of endometrial fragments; and (3) following lesion formation as a t reatment \nstrategy [45]. Nascent microvessels establish in the endometrioic lesion sites usually between \n5 and 8 days of endometrial i mplantation [45]. In our study, anti -angiogenic therapy was \nadministered two weeks following endometrial implantation  [15]. This time period is \nsufficient for vascularisation necessary for lesion establishment in the animal.  \nAdministration of four doses of 10 6 MSC-Adsflt1 following two weeks of lesion \nestablishment resulted in a highly efficient anti -angiogenesis response ; the disease \nprogression was inhibited effectively without any notable sign of new lesion growth. The \nstudy envisaged that employing MSC -Adsflt-1 gene therapy would be most suitable for the \nabrogation of nascent ectopic lesions  that may eventually help in reducing the chances of \nrecurrence of the disease . Further studies in higher animal models employing new improved \ntarget specific vectors are required to fully assess this promising s trategy in endometriosis \ncare. \nVectors used in gene therapy of endometriosis: success stories \nIn order to maximize the therapeutic effects of gene therapy and minimize toxicity on non -\ntarget tissues, specific vector designing/targeting strategies need to be implemented [46]. This \ncan be achieved by using two major approaches; transductional targeting and/or \ntranscriptional targeting.  Transductional targeting enables selective delivery of the \ntherapeutic gene at the target of interest. Transductional targeting of adenoviruses involves \nspecific modifications in the adenovirus fiber s, which includes attachment of targeting \npeptides, serotype knob switching  or fiber replacement s [47]. These modifications in the \ncapsid proteins of adenovirus would help in routing  its cell entry through receptors that are \nspecifically expressed on pathological tissues [5]. In the transcriptional targeting approach, \nthe vector may initiate non-specific gene transfer to a large number of cells; however, its \ntransgene expression is restricted specifically to the target tissue [47]. This strategy uses  \n\n10 \n \ntissue specific promoters that display preferential activity in the pathological tissues. An ideal \ntissue-specific promoter for transcriptional targeting could be defined by the selective display \nof “pathological tissue on” and “non-target tissue off” phenotype [5].  \nAdenoviral and adeno-associated vectors are being widely used for gene therapy studies [46]. \nThe advantages of recombinant adenoviruses include their eas e of propagation, limited \npathogenicity and low mutagenesis potential in humans  [5]. They are able to transfer the \ngenetic material effectively in a wide spectrum of dividing and non -dividing cells  [47]. \nHowever, the wide tropism of adenoviruses is a major limitation, since it could lead to non -\nspecific gene transfer affecting non-target cells. This is due to the recognition of the \ncoxsackie-adenovirus receptor (CAR), the cell surface receptor, by the C-terminal part of an \nadenovirus fiber protein, termed the knob [48]. CAR receptor is distributed widely over many \ncells and this poses a major drawback wherein the vector identifies even the non -target \ncells/tissues [48]. Hence, there is a need for a more specific viral vector in gene therapy based \ntransplantation studies . Othman et al developed an advanced, conditionally replicative \nadenovirus expressing tissue-specific promoters such as heparanase (Ad -heparanase-luc) [5]. \nThese targeted adenoviruses  encode luciferase reporter gene  in the E1 region under the \ntranscriptional control of the heparanase promoter. Heparanase is a heparan sulfate -specific \nglucuronidase that plays an important role in tumor cell metastasis. They cleave extracellular \nmatrices and are found to be present in endometriosis tissues [49]. Use of Ad-heparanase-luc \nresulted in an efficient removal of primary endometriotic cells obtained from ovarian \nendometrioma lesions in vitro. These viral vectors were target-specific and did not impact on \nliver tissues adversely [5].   \nAnother tissue specific promoter that has been  used for ta rgeting endometriotic cells is \nsecretory leukocyte protease inhibitor ( SLPI), which is a potent inhibitor of leukocyte serine \nproteases that protects mucosal surfaces against injury associated with inflammation  [5].   \n\n11 \n \nThe presence of transcripts of  SLPI was detected in ovarian endometrioma, peritoneal \nendometriosis, and deep recto -vaginal endometriosis; it was absent in normal ovarian tissue \nor eutopic endometrium of healthy females  [50], suggesting that SLPI  could be the likely  \npromoter for transcriptional targeting. A comparative study , however, reported a higher \nactivity for Ad -heparanse-luc vectors over Ad -SLPI-luc in endometriosis cells owing to its \nrelatively lower activity in the liver tissues [5]. Ad-heparanse-luc exhibited an “endometriosis \non, liver off” phenotype , hence, it was considered to be a promising vector for future \nendometriosis gene therapy trials [5]. \nA few reports have shown therapeutic benefits of the recombinant adenoviral and adeno -\nassociated viral vectors in the murine models of endometriosis. Adenoviral and adeno -\nassociated viral vectors carrying human angiostatin [41] and endostatin [51] genes \nrespectively could inhibit endometriosis associated angiogenesis at lesion sites.  Adenoviral \nvectors overexpressing angiostatin gene, a natural angiogenesis inhibitor,  when delivered to \nthe peritoneal cavities of mice , resulted in eradication of all the endometriotic lesions in the \nmouse model [41]. Although an effective anti-angiogenesis was observed, the study reported \na lack of target specificity. The vector infected a wide range of cells w ithin the peritoneal \ncavity.  \nRein  et al used conditionally replicative  adenoviruses  (CRADs), which  replicate  within \nand  destroy  target cells,  but  not  normal  cells , in the endometriosis models [52].  The viral \nreplication was maintained under the control of VEGF promoter and in vitro studies showed \ninduction of apoptosis in endometriotic cells.  Intra -peritoneal administration of the vector, \nhowever, resulted in a reduced VEGF promoter activity in  the liver as well as endometrium.  \nSubsequently, Paupoo et al demonstrated the promise of a modified CRAD, CRAD -S-pK7, \nwith dual advantage s of infection enhancement and promoter specificity  [53].  Polylysine \npK7 promoted the best infection enhancement of the adenoviruses whereas survivin promoter \n\n12 \n \nexhibited the highest activity in endometriotic cell lines. The CRAD -S-pK7 vector exhibited \nhigher replication rates and cell-killing efficiencies in vitro [53]. \nA suicide gene therapy approach , where an enzyme transforms a pro -drug into a toxic \nmetabolite, has been used to target  endometrial cells using adenoviral vectors  [43].  Herpes  \nsimplex  virus  thymidine  kinase  (HSV-tk)  transforms ganciclovir  (GCV)  into  ganciclovir  \ntriphosphate,  which  is toxic  for endometrial cells.  Adenoviral vectors encoding the HSV-tk \ngene (AdTK) were delivered into human endometrial  cells, which were further treated with \nGCV. This resulted in a significant level of induction of cell death.  When administered into \nmouse models of endometriosis, AdTK significantly reduced the size of the endometriotic \nlesions [43]. \nThe role of P27 protein , an important cell cycle regulatory  factor has been implicated in \nendometriosis pathogenesis [54]. P27 regulates cell cycle checkpoint at the G1 to S transition  \nstate in normal cells. Absence of P27 results in exaggerated proliferation of cells and their \ndown-regulation has been reported  in ovarian, endometrial and breast  neoplasia [54]. \nEndometriotic tissues have a dysregulated expression for cell cycle and inflammatory \nproteins and lower levels of p27kip1 protein [55]. Cells isol ated from endometriotic  tissues \nalso have lower levels of p27 kip1 compared to healthy endometrial cells  [54]. A recent study \ndemonstrated that gene therapy using Adp27 carrying the p27kip1 coding gene (Adp27EGFP), \nrestored the p27 kip1 expression, promoting a G1 cell cycle arrest and reduced cellular \nproliferation [55]. Similar studies targeting estrogen receptors, earlier described by Othman et \nal, yielded successful results using adenoviral vectors. In the in vitro system, transfer of the \ndominant negative estrogen receptor gene into human endometriotic cells resulted in  the \ninitiation of apoptosis [56].  \nConcerns yet to be addressed  \n\n13 \n \nDespite a number of promising reports highlighting the success of  gene therapy approaches  \nfor endometriosis, clinical trials remain overdue .  Although expanded MSCs have great \nproliferation and differentiation potential  in vitro, their loss of in vivo self-renewal capacity \nposes a major clinical concern. This is attributed to the extensive expansion of MSCs carried \nout during in vitro culturing, leading to the loss of their native properties when administered \nin vivo.  Thus, novel approaches are required that would allow MSCs to maintain their stem \ncell function in vivo. Likewise, recent reports on endometrial SP cells open up a new arena in \nunderstanding endometrial regeneration and its implications in several en dometrial disorders. \nWhile encouraging in vitro and in vivo results are reported, further studies are warranted to \nelucidate the full therapeutic effects of endometrial SP cells for regenerative gene therapies. \nAnother important concern is the lack of a specific set of markers in order to identify and \nisolate putative EnSC populations. Another issue is concerning the similarity of MSCs to \nother stromal cells such as fibroblasts. Thus, greater understanding of the MSC biology is \nrequired in order to redefine the complex and heterogeneous family of stromal cell  \npopulations. Safety and regulatory concerns surrounding the long term effects and fate of the \ngenetically engineered MSCs at target sites should also be addressed.  \nConclusion and future perspectives \nAddressing the above mentioned concerns would unleash t he vast clinical potential of stem \ncells for treating a range of diseases. With specific advantages associated with EnSCs, they \nare promising vehicles for cell and gene therapy -based approaches  for endometriosis and \nother disorders and are worthy of further exploration. We envisage that the next 5–10 years is \ncrucial in developing regulatory models for clini cal investigation of cell  and gene therapy  \nprotocols targeting endometriosis globally. \n \n\n14 \n \nExecutive summary \nEndometriosis: a brief overview \n Currently available therapeutic options for endometriosis are often associated with \ndisease recurrence. Thus, there is a compelling argument for developing novel \ntherapeutic approaches for the management of endometriosis. \nEndometrial stem cells (EnSCs): a promising therapeutic tool \n EnSCs have been isolated and characterized by a number of research groups and their \npromising therapeutic potential has been investigated  by several in vitro and in vivo \nstudies. \nEndometrial Stem Cells: are they flexible tools for gene therapy? \n A recent study from our laboratory explored the gene transduction potential of EnSCs. \nGenetically modified EnSCs expressed and sec reted human anti -angiogenic factor, \nsFlt-1. \nVectors used in gene therapy for endometriosis: success stories \n Recombinant a denoviral and adeno -associated vectors were successful in the gene \ntherapy of endometriosis. \nConcerns yet to be addressed  \n Clinical trials involving gene therapy of endometriosis are overdue \n Several concerns regarding clinical use of  the in vitro  expanded MSCs need to be \naddressed \n Specific set of markers for identifying and separating pure  populations of EnSCs is \nessential \n\n15 \n \n Long term effects and fate of the genetically engineered MSCs at the target sites need \nto be ascertained \nAcknowledgements/ Financial disclosure \nThe authors wish to thank Dr. S. D. Mahale, Director, NIRRH, Mumbai for providing \nnecessary facilities for research and the Post-doctoral fellowship program of the Indian \nCouncil for Medical Research, New Delhi, India  for financial support . Authors declare that \nthere is no competing financial interest. \n \n \n \n \n \n \n \n \n \n \n \n \n \n\n16 \n \nReferences \nPapers of special note have been highlighted as:  of interest;  of considerable interest \n1.  Esmaeilzadeh S, Mirabi P, Basirat Z ,  Zeinalzadeh M, Khafri  S. Association \nbetween endometriosis and hyperprolactinemia in infertile women. Iran. J. \nReprod. Med. 13: 155–160 (2015). \n2. Culley L, Law  C, Hudson  N, et al.  The social and psychological impact of \nendometriosis on women's lives: a critical narrative review.  Hum. Reprod. \nUpdate. 19: 625-639 (2013). \n3. Worley MJ Jr,  Liu S, Hua  Y, et al . Molecular changes in endometriosis -\nassociated ovarian clear cell carcinoma. Eur. J. Cancer. 51:1831-1842 (2015).  \n4. 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Gene therapy of endometriosis: \nadenovirus mediated expression of dominant negative estrogen receptor induces \napoptosis in human endometriotic cells. Fertil. Steril. 88:462–471 (2007).  \n \n \n \n\n23 \n \nFigure legend \nFigure 1: Anti -angiogenic effects of Ad sflt-1 mediated genetically modified eutopic  \nendometrial mesenchymal stem cells in SCID mouse model of endometriosis  (EM). Adsflt-1 \nviral vectors were amplified in HEK 293 cell lines at 200 plaque forming unit  (pfu)/cell. The \nvirions were later released from host HEK cells by repeated freeze thaw te chnique. Eutopic \nMSCs were infected with purified Adsflt-1 viral particles at a multiplicity of infection (MOI)  \nof 2000:1. Expression and release of sFlt -1 by transduced eutopic MSCs were confirmed by \nflow cytometry and western blotting analysis. SCID mouse model  of endometriosis was  \ncreated by subcutaneous endometrial implantation. Following two weeks of endometrial \nimplantation, four doses of 10 6 MSC-Adsflt-1 were administered via tai l vein. Animals were \nsacrificed after three weeks of initiation of t he study and monitored for signs of lesion \ndevelopment, invasion, migration and angiogenesis. \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n\n24","source_license":"CC0","license_restricted":false}