human pluripotent stem cells ( hpscs ) , including human embryonic stem cells ( hescs ) and human induced pluripotent stem cells ( hipscs ) , are indefinitely self - renewing and pluripotent . these properties make hpscs a promising platform for cell therapy , as well as a possible alternative to organ transplantation . consequently , hpscs have attracted increasing attention from scientists in the arenas of basic and applied biomedical research . hescs have been employed in clinical trials involving human subjects in north america , europe , and other regions of the world , and a clinical study using hipscs has been initiated in japan.2 , 3 however , the utility of hpscs in regenerative medicine can not be generalized on the basis of this small number of early clinical studies ; instead , their applicability must be evaluated on an organ - specific basis . concerns regarding the safety of hpscs , in particular their tumorigenic potential , must be fully addressed before the hpsc technologies can be widely applied in clinical regenerative medicine . transplanted cells containing undifferentiated hpscs may give rise to benign teratomas or malignant tumors ( figure  1 ) . here we review the tumorigenic potential of hpscs , its impact on the safety of regenerative medicine , and current technological limitations on the prevention of tumor formation . we also discuss new approaches for directly eliminating the tumorigenic potential of hpscs , with a focus on the current status and future prospects of viral vector - based techniques ( figure  2 ) . safety should be the primary consideration in clinical and commercial application of innovative pharmaceutical and medical products . agents with a high carcinogenic potential , no matter how beneficial they might be , must be eliminated from the development pipeline at an early stage . in the arena of regenerative medicine , while stem cells are expected to provide a powerful means for innovative therapy that outdates conventional medicine , they pose a significant risk of tumorigenesis . in practical terms , the unintended negative consequences of genetically manipulating stem cells were demonstrated in a clinical trial of ex  vivo hematopoietic stem cell gene therapy in patients with x - linked severe combined immunodeficiency ; this study provided biologists and clinicians with several important lessons about stem cell tumor formation . a high incidence of leukemia was documented in treated patients , although nonclinical studies had concluded otherwise . nonclinical studies involving experimental animals may be useful for predicting the levels of tumor suppression in cell transplant therapy , but they can never ensure that the therapy is clinically safe and free from tumor risk in humans . in that study , the therapeutic gene (  subunit of the interleukin-2 receptor gene ) the vector also activated the locus associated with a known t  cell oncogene , lim domain only2 ( lmo2 ) gene , which clinically manifested a few years after treatment . due to its selective growth advantage , a single neoplastic stem cell can lead to the formation of a clinically significant cancer . therefore , to ensure clinical safety , such tumorigenic cells must be completely eradicated from the transplanted cell population . due to the fundamental biological properties of hpscs , regenerative medicine technologies using hpscs are associated with a higher risk of tumor generation than those using tissue stem cells ( figure  1 ) . first , long - term culture in  vitro makes undifferentiated hpscs susceptible to mutations due to their genetic instability . consequently , even if a master cell bank ( mcb ) is established from a clone whose genome has been completely sequenced , its cells may undergo mutations during the course of large - scale culture for clinical application . it is not practical to sequence the genome of individual cells in the final hpsc - derived transplant cell products . in addition , researchers have recently reported that , due to premature termination of reprogramming , hipscs undergo transformation via altered epigenetic regulation in the absence of genetic changes . to overcome the risk of tumorigenesis , researchers have tried to reduce the presence of undifferentiated hpscs and tumorigenic cells in the cell culture . those methods include improvements in the cell culture and reprogramming methods , establishment of safe hpsc mcbs through gene expression analysis and genome - wide sequencing , and development of efficient methods for inducing differentiation ( figure  1).4 , 7 notably , these conventional methods aim to suppress tumorigenesis indirectly . given the possible presence of remnants with tumorigenic potential and the selective growth advantage of cancer cells , however , these techniques can not fully suppress tumor development , although they can effectively decrease the chance of , or extend the time to , tumorigenesis . therefore , in order to achieve a reliable hpsc - based regenerative medicine , a completely new and innovative strategy for directly and specifically killing or eliminating tumorigenic cells is urgently required . because this review discusses novel targeted techniques for destroying or removing tumorigenic cells , with a focus on viral vector - based approaches , we summarize the limitations of non - viral vectors and the characteristic features of representative viral vectors for hpsc transduction . non - viral gene transfection and expression techniques applied to hpscs may be broadly categorized as physical ( e.g. , electroporation ) or chemical ( e.g. , lipofection ) . for example , depending on the size of genes to be transferred and the type of hpsc line , non - viral techniques often can not achieve satisfactory transfection efficiencies . in addition , these techniques achieve short - lived gene expression that lasts for a few days , and they often lead to the death of hpscs due to their susceptibility to transfection - induced cellular stress . by contrast , viral vectors can achieve high transduction efficiency and long - term gene expression , and they are , therefore , quite useful for genetic hpsc manipulation , although their production is technically demanding . the common types of vectors used for hpsc engineering are single - stranded rna retroviruses and lentiviruses , which can be generated relatively easily.8 , 9 such vectors insert viral genes into the host dna , theoretically enabling long - term expression of the inserted genes . however , these viral vectors have the noteworthy disadvantage that exogenous genes are silenced at a high frequency in undifferentiated pluripotent stem cells , including hpscs , when viral long terminal repeats ( ltrs ) are used as their promoters.10 , 11 this problem can be circumvented by using self - inactivating vectors that lack viral enhancers / promoters in the 3 ltr ; such vectors allow for exogenous promoters to control transgenic expression . in addition , pseudotyping with vesicular stomatitis virus g glycoprotein enables vector concentration , which , in combination with the broad tropism of the glycoprotein , increases transduction efficiency . viral vectors that integrate exogenous genes into the host chromosome may possibly pose significant safety risks . the retrovirus - mediated stem cell gene therapy mentioned earlier caused a high incidence of leukemia in treated patients . therefore , an mcb should probably be established from hpsc clones that have been shown to carry transgenes at safe genomic sites . the application of double - stranded dna adenoviral vectors to pluripotent stem cells , including hpscs , has been studied by a very small number of researchers , including the authors of this review.10 , 13 , 14 because adenoviral vectors can be produced at much higher titers ( 10 infectious units / ml ) than retroviral and lentiviral vectors ( 10 infectious units / ml ) , the in  vitro transduction efficiency of adenoviral vectors can be increased to nearly 100% by elevating the moi . in addition , adenoviral dna ( including any exogenous genes ) does not integrate into the host chromosome but persists as an episome in the nucleus , allowing stable long - term transgene expression under the control of exogenous promoters.10 , 15 because the host chromosome remains intact , the episomal nature of the adenoviral vector confers a significant advantage in clinical safety . it should be noted that the percentage of host hpscs transduced with the episomal vector decreases rapidly with active cell division . accurate knowledge and precise command of the properties of adenoviral vectors should facilitate discovery of new modalities for eradicating tumorigenic cells present in hpsc cultures . suicide genes , a group of genes whose products induce cell death , have been extensively investigated in cancer gene therapy , and they have been used to kill or remove undifferentiated tumorigenic cells . one approach is based on a well - studied gene therapy involving the herpes simplex virus thymidine kinase ( hsv - tk ) and ganciclovir ( gcv).16 , 17 , 18 gcv , a nontoxic prodrug , is phosphorylated by intracellular hsv - tk and ultimately converted to gcv - triphosphate , which potently induces cell death by inhibiting dna replication ( figure  3 ) . lentivirus - transduced and undifferentiated hpscs expressing the hsv - tk gene are effectively eliminated in  vitro in the presence of gcv.19 , 20 in addition , in  vivo teratomas derived from hsv - tk - transduced hpscs shrink following the administration of gcv . hsv - tk  + gcv therapy has the additional advantage of a bystander effect , in which the therapeutic effect of the drug is manifested in genetically unmodified cancer cells adjacent to the modified ones.16 , 17 , 18 however , it remains unclear to what extent the bystander effect occurs in hpscs . the potential of the hsv - tk  + gcv method to kill non - hsv - tk - transduced cells or hsv - tk - transduced cells whose hsv - tk expression has been silenced represents an additional advantage of this approach . previous research using adoptive t  cells and mesenchymal stromal cells showed that the inducible caspase 9 ( ic9 ) protein , mediated by a small molecule dimerizer , activates caspase 3 , which in turn eliminates damaged or unwanted cells ( figure  3 ) . recent work showed that the ic9 suicide system has the potential to kill tumorigenic cells and prevent teratoma formation in hpsc populations . lentivirus - transduced hipscs expressing the ic9 gene mostly died by apoptosis within 24  hr of dimerizer treatment , and teratomas derived from ic9-expressing cells shrank in  vivo following the administration of the dimerizer . in addition , hipscs established from antigen - specific t  cells and transduced by a lentivirus vector expressing the ic9 gene died by apoptosis after dimerizer treatment . their findings suggested that the ic9 gene acts as a safety switch that eliminates tumor cells that develop over the course of clinical application , thereby improving the outcome of hipsc - derived t  cell therapy . it should be noted , however , that these observations only show that this system can kill tumorigenic cells , prevent teratoma formation , and provide a safeguard against aberrant cells . no study to date has determined the level of suicide gene expression necessary to completely destroy all tumorigenic cells contained in a given hpsc population . in addition , it remains unknown whether the expression of suicide genes damages the differentiated cells of interest . constitutively active promoters and the promoter of the pluripotency marker nanog have been used to regulate suicide gene expression.19 , 21 , 22 however , little is known about the degrees of effectiveness of other promoters , as well as their specificity for the undifferentiated state , using this system . in our own research on cancer gene therapy , we have identified an optimal therapeutic expression level for suicide gene , above which inducer ( gcv)-independent nonspecific cell death occurs . when suicide genes are expressed above the optimal expression level in animal models , they tend to experience more severe adverse reactions , and the therapeutic effect of the suicide genes does not increase . our previous work suggested the need to identify promoters that activate only undifferentiated cells ( not the differentiated target cells ) in a highly specific manner and induce optimal levels of suicide genes for maximum tumoricidal effects . to achieve this goal , we developed a tumorigenic cell - targeting lentiviral vector ( tc - lv ) platform that can incorporate promoter and fluorescent protein genes in the recombination cassette . we have used this platform to efficiently and simultaneously generate a variety of tc - lvs that encode different types of promoters , and these vectors are currently being used to identify the promoters that most effectively kill tumorigenic cells ( k.i . , unpublished data ) . oncolytic virotherapy , using viruses engineered to selectively replicate in and kill tumor cells , has emerged as an innovative and promising strategy for cancer treatment.23 , 24 a wide variety of oncolytic viruses have been developed that take advantage of the properties of the original viral species . we developed our unique vector by further modification of a conditionally replicating adenovirus ( cra ) , which was derived from a human adenovirus . this cra , designated a conditionally replicating adenovirus regulated with multiple tumor - specific factors ( m - cra ) , is regulated by multiple tumor - specific factors ( figure  4 ) . we devised a set of procedures to incorporate into m - cra three independently constructed regulatory clusters that encode viral replication , therapeutic , and adenoviral backbone genes . we constructed a variety of m - cras expressing different properties , and we analyzed their therapeutic effect and cancer specificity ( i.e. ,  safety ) . the most favorable results were obtained with surv.m - cras , whose replication is controlled by the survivin promoter ( figure  4).25 , 26 survivin ( birc5 ) , an inhibitor of apoptotic proteins , is overexpressed in most cancers . its expression levels correlate with cancer prognosis , and survivin promoter activity is upregulated in cancer cells due to aberrant transcriptional activation . comparison of the viral properties and therapeutic potentials of surv.m - cra and telomerase reverse transcriptase ( tert)-responsive m - cra ( tert.m - cra ) revealed that surv.m - cra was safer and more effective . another group employed a similar tert - responsive cra in a human cancer trial and reported a positive local effect and safety . surv.m - cra demonstrated elevated effectiveness against cancer stem cells , which are often resistant to conventional anticancer medications and radiotherapy . we are currently implementing a first - in - human investigator - initiated trial of surv.m - cra for the treatment of refractory malignant bone and soft tissue tumors . we recently developed a method for employing oncolytic viruses to selectively and efficiently kill undifferentiated tumorigenic cells in hpsc culture using the surv.m - cra and tert.m - cra models . first , survivin , long considered a cancer - specific promoter , was unexpectedly more active in undifferentiated hpscs than the tert promoter , but it was minimally active in differentiated hpscs . second , both m - cras exhibited efficient viral replication and cytocidal potential in undifferentiated hpscs ( with surv.m - cra more potent than tert.m - cra ) , but not in co - cultured differentiated normal cells . third , pre - infection of hpscs with either m - cra prevented teratoma formation in  vivo . thus , our m - cra platform technology and surv.m - cra product , originally developed for cancer treatment , provide an effective means for abolishing undifferentiated tumorigenic hpscs and producing tumor - free hpsc populations . on the other hand , because transcriptional regulation of a viral gene by a cancer - specific promoter failed to achieve cancer - specific viral replication in certain types of viruses , not all oncolytic viruses are applicable to , or effective for , selective eradication of tumorigenic hpscs . for instance , representative cancer - specific promoters , including the survivin and the tert promoters that functioned well in surv.m - cras and tert.m - cras as well as in plasmids , drastically lost their cancer - specific potential in herpes simplex viruses.30 , 31 although many methods for inducing targeted differentiation have been reported in the literature , it is not currently feasible to obtain the target cells with 100% efficiency . consequently , the transplant cell population contains a certain amount of unwanted types of differentiated and undifferentiated cells . to overcome this technical limitation , it is necessary to devise a purification method that isolates target cells from a heterogeneous culture . currently , however , no surface antigen markers are available that could assist in identifying cardiomyocytes or many other types of cells . for many years , the only solution to this problem was to establish hpscs that stably expressed reporter genes under the control of a tissue - specific gene promoter . this approach poses multiple challenges ; it is time- and resource consuming to establish such stable cell lines , and the dependence of fluorescence intensity on target gene promoter activity means that fluorescence visualization can not be achieved when activity is low . we developed a technique for ensuring robust fluorescence protein expression , namely , the adenoviral conditional targeting in stem cell ( act - sc ) method ( figure  5 ) . this method employs adenoviral vectors carrying two dna constructs encoding the cre protein and an expression - switching unit . the use of the adenoviral vector enables straightforward transduction of genes of interest into hpscs by simply mixing the vector cocktail with the hpsc culture , even during the process of induction . vectors also facilitate stable expression of the virally transduced genes due to their episomal nature , as explained earlier . even if the cre expression level is extremely low , it excises the loxp - flanked stuffer gene only in the target cell , leading to transcription of the downstream fluorescent gene under the strong constitutively active promoter . using the nkx2.5 promoter , we successfully identified and purified primitive cells of the cardiac lineage for  the first time . we also succeeded in separating mature cardiomyocytes from mouse embryonic stem cells using the sarcomeric -myosin heavy chain promoter . in addition , we showed that act - sc is quite useful for isolating hpsc - derived target cells , and we have identified the optimal experimental conditions for doing so ( k.m . , unpublished data ) . high - purity isolation of target cells is critical for improving the effectiveness and safety of the hpsc therapy ( figure  2 ) . in addition to the aforementioned methods for abolishing tumorigenic cells , act - sc will play an important role in future clinical applications of hpscs as a means to increase the homogeneity of transplanted cells . on the other hand , one potentially unfavorable feature of act - sc for clinical application is the persistence of remnant adenoviral genomes in the transplanted hpsc - derived target cells , which may induce some degree of cellular immunity against the transplanted cells . nevertheless , this issue could be solved by optimization of the clinical conditions , e.g. , the use of adenoviral vectors at an adequate moi and subsequent culture to eliminate episomal adenoviral genomes . here we briefly describe several methods for eradicating tumorigenic cells that take advantage of the biological properties of undifferentiated cells ( figure  2 ) . one group of researchers showed that prolonged methionine deprivation induces cell death more readily in undifferentiated than differentiated cells , because undifferentiated cells demand more methionine than differentiated cells . another group of researchers developed a recombinant lectin - toxin fusion protein that combines an hpsc - specific lectin probe , rbc2lcn , with the catalytic domain of pseudomonas aeruginosa exotoxin a. this fusion protein has the potential to completely eliminate hpscs . yet another group synthesized a molecular switch for microrna-302a-5p , a molecule that is highly and specifically expressed in undifferentiated hpscs but gradually decreases to basal levels during differentiation . the early success of these techniques warrants further detailed investigation of their tumor selectivities and impacts on differentiated target cells . it should be noted that these techniques are generally applicable to undifferentiated pre - transplant cells , but they can not address post - transplant cell tumorigenesis or cancer development ( figure  2 ) . in this regard , we believe that viral transduction of suicide genes has a clinical advantage over other methods , because such genes serve as a safeguard against possible tumorigenesis . safe and robust techniques to prevent hpsc tumorigenesis are indispensable for the advancement of regenerative medicine . in addition to existing indirect methods for reducing tumorigenic cell contamination , new techniques should be developed that can directly , securely , and specifically eradicate tumorigenic cells . the innovative viral vector - based technologies reviewed in this article promise to have far - reaching impacts that supersede conventional approaches . future development and optimization of these and other new technologies will help to overcome the problems of hpsc tumorigenesis in the context of regenerative medicine .
human pluripotent stem cells ( hpscs ) are a promising source of regenerative material for clinical applications . however , hpsc transplant therapies pose the risk of teratoma formation and malignant transformation of undifferentiated remnants . 
 these problems underscore the importance of developing technologies that completely prevent tumorigenesis to ensure safe clinical application . 
 research to date has contributed to establishing safe hpsc lines , improving the efficiency of differentiation induction , and indirectly ensuring the safety of products . despite such efforts , guaranteeing the clinical safety of regenerative medicine products 
 remains a key challenge . 
 given the intrinsic genome instability of hpscs , selective growth advantage of cancer cells , and lessons learned through failures in previous attempts at hematopoietic stem cell gene therapy , conventional strategies are unlikely to completely overcome issues related to hpsc tumorigenesis . 
 researchers have recently embarked on studies aimed at locating and directly treating hpsc - derived tumorigenic cells . 
 in particular , novel approaches to directly killing tumorigenic cells by transduction of suicide genes and oncolytic viruses are expected to improve the safety of hpsc - based therapy . 
 this article discusses the current status and future perspectives of methods aimed at directly eradicating undifferentiated tumorigenic hpscs , with a focus on viral vector transduction .