MAPK activation drives male and female mouse teratocarcinomas from late primordial germ cells.

Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior.

Selective elimination of pluripotent stem cells by PIKfyve specific inhibitors.

Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.

The foundational framework of tumors: Gametogenesis, p53, and cancer.

The completion-of-tumor hypothesis involved in the dynamic interplay between the initiating oncogenic event and progression is essential to better recognize the foundational framework of tumors. Here we review and extend the gametogenesis-related hypothesis of tumors, because high embryonic/germ cell traits are common in tumors. The century-old gametogenesis-related hypothesis of tumors postulated that tumors arise from displaced/activated trophoblasts, displaced (lost) germ cells, and the reprogramming/reactivation of gametogenic program in somatic cells. Early primordial germ cells (PGCs), embryonic stem (ES) cells, embryonic germ cells (EGCs), and pre-implantation embryos at the stage from two-cell stage to blastocysts originating from fertilization or parthenogenesis have the potential to develop teratomas/teratocarcinomas. In addition, the teratomas/teratocarcinomas/germ cells occur in gonads and extra-gonads. Undoubtedly, the findings provide strong support for the hypothesis. However, it was thought that these tumor types were an exception rather than verification. In fact, there are extensive similarities between somatic tumor types and embryonic/germ cell development, such as antigens, migration, invasion, and immune escape. It was documented that embryonic/germ cell genes play crucial roles in tumor behaviors, e.g. tumor initiation and metastasis. Of note, embryonic/germ cell-like tumor cells at different developmental stages including PGC and oocyte to the early embryo-like stage were identified in diverse tumor types by our group. These embryonic/germ cell-like cancer cells resemble the natural embryonic/germ cells in morphology, gene expression, the capability of teratoma formation, and the ability to undergo the process of oocyte maturation and parthenogenesis. These embryonic/germ cell-like cancer cells are derived from somatic cells and contribute to tumor formation, metastasis, and drug resistance, establishing asexual meiotic embryonic life cycle. p53 inhibits the reactivation of embryonic/germ cell state in somatic cells and oocyte-like cell maturation. Based on earlier and our recent studies, we propose a novel model to complete the gametogenesis-related hypothesis of tumors, which can be applied to certain somatic tumors. That is, tumors tend to establish a somatic asexual meiotic embryonic cycle through the activation of somatic female gametogenesis and parthenogenesis in somatic tumor cells during the tumor progression, thus passing on corresponding embryonic/germ cell traits leading to the malignant behaviors and enhancing the cells' independence. This concept may be instrumental to better understand the nature and evolution of tumors. We rationalize that targeting the key events of somatic pregnancy is likely a better therapeutic strategy for cancer treatment than directly targeting cell mitotic proliferation, especially for those tumors with p53 inactivation.

A Comprehensive Analysis of Human Endogenous Retroviruses HERV-K (HML.2) from Teratocarcinoma Cell Lines and Detection of Viral Cargo in Microvesicles.

About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles-MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.

Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC.

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.

Murine Teratocarcinoma-Derived Neuronal Cultures.

This chapter describes the culture and propagation of murine embryonic stem cells, F9 and P19, and strategies for differentiation of these stem cells into neurons. Additional techniques are described for obtaining enriched populations of mature neurons from P19 cells and differentiation of F9 cells into serotonergic or catecholaminergic neurons. The protocols described herein can be used for dissection of the pathways such as gliogenesis and neurogenesis that are involved in differentiation of pluripotent stem cells such as F9 and P19 into glial cells or terminally differentiated neurons.

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth.

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.

LINC00324 facilitates cell proliferation through competing for miR­214­5p in immature ovarian teratocarcinoma.

Immature ovarian teratocarcinoma (IOT) is a rare and malignant type of ovarian teratoma, and the molecular mechanisms underlying the pathogenesis and malignant phenotype of IOT remain uncharacterized. The present study examined a long non­coding RNA (lncRNA), long­chain intergenic non­coding RNA324 (LINC00324), which may serve a crucial role in pathogenesis of IOT. According to the results, LINC00324 was upregulated in IOT tissues and cells, as determined by reverse transcription­quantitative PCR, and its depletion impaired cell proliferation ability and improved cell apoptosis ability in IOT. Furthermore, LINC00324 acted as a miR­214­5p sponge to derepress cyclin dependent kinase 6 (CDK6), cyclin D1 (CCND1), murine double minute homolog 2 (MDM2), and mouse double minute 4 (MDM4) expression, thus increasing IOT cell proliferation and repressing apoptosis. Taken together, these results demonstrated that LINC00324 could serve as a competing endogenous RNA to facilitate IOT cell proliferation by regulation of miR­214­5p­CDK6/CCND1/MDM2/MDM4 network, which possibly provide a novel therapeutic target for IOT.

TGFß Family Signaling Pathways in Pluripotent and Teratocarcinoma Stem Cells' Fate Decisions: Balancing Between Self-Renewal, Differentiation, and Cancer.

The transforming growth factor-ß (TGFß) family factors induce pleiotropic effects and are involved in the regulation of most normal and pathological cellular processes. The activity of different branches of the TGFß family signaling pathways and their interplay with other signaling pathways govern the fine regulation of the self-renewal, differentiation onset and specialization of pluripotent stem cells in various cell derivatives. TGFß family signaling pathways play a pivotal role in balancing basic cellular processes in pluripotent stem cells and their derivatives, although disturbances in their genome integrity induce the rearrangements of signaling pathways and lead to functional impairments and malignant transformation into cancer stem cells. Therefore, the identification of critical nodes and targets in the regulatory cascades of TGFß family factors and other signaling pathways, and analysis of the rearrangements of the signal regulatory network during stem cell state transitions and interconversions, are key issues for understanding the fundamental mechanisms of both stem cell biology and cancer initiation and progression, as well as for clinical applications. This review summarizes recent advances in our understanding of TGFß family functions in naїve and primed pluripotent stem cells and discusses how these pathways are involved in perturbations in the signaling network of malignant teratocarcinoma stem cells with impaired differentiation potential.

Analysis of Human Stem Cell Transcription Factors.

Transcription factors NANOG, OCT4, SOX2, and NESTIN are expressed in both human embryonic stem cells (hESCs) and cancer stem cells and they play a crucial role in maintaining characteristics of stemness such as self-renewal and pluripotency. This article evaluates the expression of variants of the main stem cell-specific transcription factors NANOG and OCT4 critically and accurately with specific primers designed for identifying the most important variants that maintain stemness. We have examined four variants of NANOG along with a processed pseudogene and seven variants of OCT4 in human teratocarcinoma cell lines (NTERA2D1, SuSa, GCT-27, and 833KE), hESCs, and ovarian cancer cells by reverse transcriptase-polymerase chain reaction. In addition, we have examined their expression in NTERA2D1 cells on differentiation with all-trans-retinoic-acid. We show that NANOG1 is expressed in all teratocarcinoma cells and can be distinguished from NANOGP8, which is an expressed pseudogene. NANOG2 was not expressed in any of the cell lines, including ESCs. OCT4A was expressed in all cells, whereas the variant OCT4B-variant 3 was expressed only in NTERA2D1 cells. On differentiation of NTERA2D1 with retinoic acid, only NANOGP8 and OCT4A were expressed. In ovarian cancer cells, only 3/6 expressed NANOG1 and OCT4A. All malignant cells from patients with ovarian cancer (N = 6) expressed NANOG1 and OCT4A. These results demonstrate the necessity to precisely evaluate the expression of stem cell transcription factors when defining stemness.

The HERV-K accessory protein Np9 controls viability and migration of teratocarcinoma cells.

Human endogenous retroviruses are remnants of ancient germline infections that make up approximately 8% of the modern human genome. The HERV-K (HML-2) family is one of the most recent entrants into the human germline, these viruses appear to be transcriptionally active, and HERV-K viral like particles (VLPs) are found in cell lines from a number of human malignancies. HERV-K VLPs were first found to be produced in teratocarcinoma cell lines, and since then teratocarcinoma has been thought of as the classical model for HERV-Ks, with the NCCIT teratocarcinoma cell line particularly known to produce VLPs. Treatment for teratocarcinoma has progressed since its discovery, with improved prognosis for patients. Since the introduction of platinum based therapy, first year survival has greatly improved even with disseminated disease; however, it is estimated that 20% to 30% of patients present with metastatic germ cell tumor relapse following initial treatments. Also, the toxicity associated with the use of chemotherapeutic agents used to treat germ cell tumors is still a major concern. In this study, we show that the depletion of the HERV-K accessory protein Np9 increases the sensitivity of NCCIT teratocarcinoma cells to bleomycin and cisplatin. While decreasing the expression of Np9 had only a modest effect on the baseline viability of the cells, the reduced expression of Np9 increased the sensitivity of the teratocarcinoma cells to environmental (serum starvation) and chemical (chemotherapeutic) stresses. Np9 is also essential to the migration of NCCIT teratocarcinoma cells: in a wound closure assay, reduced expression of Np9 resulted in cells migrating into the wound at a slower rate, whereas reintroduction of Np9 resulted in NCCIT cells migrating back into the wound in a manner similar to the control. These findings support the implication that the HERV-K accessory protein Np9 has oncogenic potential.

Peroxisome proliferator-activated receptor alpha accelerates neuronal differentiation and this might involve the mitogen-activated protein kinase pathway.

Activation of peroxisome proliferator-activated receptor alpha (PPARα) has been reported to modulate cell proliferation, migration, and differentiation in astrocytes. In this study, we used a retinoic acid (RA)-induced differentiation model of NTERA-2/clone D1 (NT2) cells to explore the functional significance of PPARα in neuronal differentiation. We found that activating PPARα by Wy14643 accelerated neuronal differentiation via regulating the expression of neuronal markers. RT-PCR assays showed a significant increase in NeuroD expression and a decrease in nestin expression in cells treated concomitantly with RA and Wy14643 for 2 days compared to the levels in cells treated with RA alone. Expression of MAP2 protein, a mature neuronal marker, was markedly upregulated at day 10 of Wy14643 treatment, which was maintained after 21 days of neuronal formation. Corresponding to the changes in MAP2 expression, the expression of Cdk5 was upregulated with Wy14643 exposure from day 10 to day 21. Moreover, cells treated with Wy14643 displayed higher expression levels of phospho-ERK and phospho-p38 in the differentiation process than cell treated with RA alone. These results indicated that activation of PPARα accelerated neuronal differentiation through upregulating the expression of NeuroD, MAP2, and Cdk5 and downregulating the expression of nestin. MAPK signals, ERK and p38, might contribute to the accelerated differentiation process. These findings suggest that PPARα plays a role in regulating neuronal differentiation and may be beneficial for functional recovery from neurological disorders.

LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.

In Vitro Anticancer Effect of Gedunin on Human Teratocarcinomal (NTERA-2) Cancer Stem-Like Cells.

Gedunin is one of the major compounds found in the neem tree (Azadirachta indica). In the present study, antiproliferative potential of gedunin was evaluated in human embryonal carcinoma cells (NTERA-2, a cancer stem cell model) and peripheral blood mononuclear cells (PBMCs), using Sulforhodamine (SRB) and WST-1 assays, respectively. The effects of gedunin on expression of heat shock protein 90 (HSP90), its cochaperone Cdc37, and HSP client proteins (AKT, ErbB2, and HSF1) were evaluated by real-time PCR. Effects of gedunin on apoptosis were evaluated by (a) apoptosis associated morphological changes, (b) caspase 3/7 expression, (c) DNA fragmentation, (d) TUNEL assay, and (e) real-time PCR of apoptosis related genes (Bax, p53, and survivin). Gedunin showed a promising antiproliferative effect in NTERA-2 cells with IC50 values of 14.59, 8.49, and 6.55 µg/mL at 24, 48, and 72 h after incubations, respectively, while exerting a minimal effect on PBMCs. Expression of HSP90, its client proteins, and survivin was inhibited and Bax and p53 were upregulated by gedunin. Apoptosis related morphological changes, DNA fragmentation, and increased caspase 3/7 activities confirmed the proapoptotic effects of gedunin. Collectively, results indicate that gedunin may be a good drug lead for treatment of chemo and radiotherapy resistant cancer stem cells.

Frizzled gene expression and negative regulation of canonical WNT-ß-catenin signaling in mouse F9 teratocarcinoma cells.

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT-ß-catenin pathway. The upregulation of Wnt6 and activation of ß-catenin-TCF-LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT-ß-catenin signaling, thereby allowing PE cells to differentiate.

Overexpression of Cathepsin E Interferes with Neuronal Differentiation of P19 Embryonal Teratocarcinoma Cells by Degradation of N-cadherin.

Cathepsin E (CatE), an aspartic protease, has a limited distribution in certain cell types such as gastric cells. CatE is not detectable in the normal brain, whereas it is increasingly expressed in damaged neurons and activated microglia of the pathological brain. Neurons expressing high levels of CatE showed apparent morphological changes, including a marked shrinkage of the cytoplasmic region and beading of neurites, suggesting neuronal damage. The intracellular level of CatE in neurons is strictly regulated at both transcriptional and translational levels. Although the up-regulation of CatE may cause pathological changes in neurons, little information is available about the precise outcome of the increased expression of CatE in neurons. In this study, we have attempted to clarify the outcome of up-regulated CatE gene expression in neurons using the P19 cell neuronal differentiation after the overexpression of CatE. We unexpectedly found that the overexpression of CatE interfered with neuronal differentiation of P19 cells through an impairment of cell aggregate formation. Pepstatin A, an aspartic protease inhibitor, restored the impaired cell aggregation of P19/CatE cells. The small number of P19 cells differentiated into neurons had abnormal morphology characterized by their fusiform cell bodies with short processes. Furthermore, CatE proteolytically cleaved the extracellular domain of N-cadherin. These observations suggest that the overexpression of CatE interferes with neuronal differentiation of P19 cells through an impairment of cell aggregate formation, possibly through proteolytic degradation of N-cadherin.

Correlative Studies Unravelling the Possible Mechanism of Cell Death in Tideglusib-Treated Human Ovarian Teratocarcinoma-Derived PA-1 Cells.

This study aims to unravel the use of GSK-3 inhibitors as viable apoptotic inducers for teratocarcinoma-derived ovarian PA-1 cells. MTT assay was carried out to assess inhibitory concentrations of LiCl and TDG. AO/EB staining and Hoechst 33258 staining were employed to assess the damage. Mitochondrial membrane potential (ΔΨm) and ROS generation were assessed with IC50 concentrations of LiCl and TDG. Tumor-related genes (p53, p21, IL-8, TNF-α, MMP-2, Fas-L, Cox-2, and caspase-3) were assessed with 1/4 IC50, 1/2 IC50, IC50 concentrations by semi-quantitative RT- PCR. Cell cycle analysis was performed with IC50 concentration of LiCl and TDG. Western blot analysis was performed for caspase-3, caspase-7, caspase-9, PARP to estimate the possible damage induced by GSK-3 inhibitors and regulation of GSK-3ß, pGSK-3ß, Cox-2. GSK-3 inhibitors demonstrated a concentration and time-dependent reduction in cell viability, exhibiting significant ROS generation and reduced ΔΨm at their IC50 values. Substantial concentration-dependent gene expression changes with significant upregulation of P21, Cox-2, TNF-α, caspase-3, Fas-L were observed. Protein expression of caspase-3 caspase-7, caspase-9, PARP exhibited significant cleavage in LiCl and TDG-treated cells. Protein expression of Cox-2 was significantly increased in IC50 concentration of TDG. Cell cycle analysis showed significant accumulation of cells at sub-G0-G1.

Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma.

Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration.

UNLABELLED: Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. SIGNIFICANCE: Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature ß cell phenotype would lead to safe islet replacement therapy for diabetes.

Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor.

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.