Teratoma Growth Research Articles

Challenges in the diagnosis of growing teratoma syndrome: two case reports

Growing teratoma syndrome (GTS) is reported to have an incidence of 1.9–7.6% among all germ cell tumours (GCT) and is often mistaken for tumor progression. There are no absolute signs or symptoms of GTS but it can be suspected based on the following criteria: the normalization of tumour markers, the enlargement of the mass and the presence in the tumour of mature teratoma cells alone. A delayed tumor resection in GTS can result in higher surgical risks and/or the infeasibility of complete resection. In 72–83% of cases, incomplete resection leads to relapses in the form of mature teratoma growth. A repeat biopsy is recommended in all patients with suspected GTS so that to avoid chemotherapy that would be ineffective in this disease. The article reports two clinical cases of GTS illustrating challenges inherent in the diagnosis of this syndrome. In each case, the parents gave consent to the use of their child's data, including photographs, for research purposes and in publications.

Teratoma Growth Retardation by HDACi Treatment of the Tumor Embryonal Source.

Simple SummaryTesticular germ cell tumors are the most common neoplasms in young male populations, with a rising incidence. Among them, teratomas may often be very aggressive and resistant to therapy. Our aim was to investigate the impact of two potential anti-tumor epigenetic drugs (Valproate and Trichostatin A) in a mammalian model of teratoma development from an early trilaminar mouse embryo. Both drugs applied to the embryonic tissue had a significant negative impact on the teratoma growth in a three-dimensional in vitro culture. However, Trichostatin A did not diminish some potentially dangerous features of teratomas in contrast to Valproate. This research is an original contribution to the basic knowledge of the origin and development of teratomas. Such knowledge is necessary for envisioning therapeutic strategies against human testicular tumors.Among testicular germ cell tumors, teratomas may often be very aggressive and therapy-resistant. Our aim was to investigate the impact of histone deacetylase inhibitors (HDACi) on the in vitro growth of experimental mouse teratoma by treating their embryonic source, the embryo-proper, composed only of the three germ layers. The growth of teratomas was measured for seven days, and histopathological analysis, IHC/morphometry quantification, gene enrichment analysis, and qPCR analysis on a selected panel of pluripotency and early differentiation genes followed. For the first time, within teratomas, we histopathologically assessed the undifferentiated component containing cancer stem cell-like cells (CSCLCs) and differentiated components containing numerous lymphocytes. Mitotic indices were higher than apoptotic indices in both components. Both HDACi treatments of the embryos-proper significantly reduced teratoma growth, although this could be related neither to apoptosis nor proliferation. Trichostatin A increased the amount of CSCLCs, and upregulated the mRNA expression of pluripotency/stemness genes as well as differentiation genes, e.g., T and Eomes. Valproate decreased the amount of CSCLCs, and downregulated the expressions of pluripotency/stemness and differentiation genes. In conclusion, both HDACi treatments diminished the inherent tumorigenic growth potential of the tumor embryonal source, although Trichostatin A did not diminish the potentially dangerous expression of cancer-related genes and the amount of CSCLC.

Embryo-derived teratoma invitro biological system reveals antitumor and embryotoxic activity of valproate.

Antiepileptic/teratogen valproate (VPA) is a histone deacetylase inhibitor/epigenetic drug proposed for the antitumor therapy where it is generally crucial to target poorly or undifferentiated cells to prevent a recurrence. Transplanted rodent gastrulating embryos‐proper (primitive streak and three germ layers) are the source of teratoma/teratocarcinoma tumors. Human primitive‐streak remnants develop sacrococcygeal teratomas that may recur even when benign (well differentiated). To screen for unknown VPA impact on teratoma‐type tumors, we used original 2‐week embryo‐derived teratoma in vitro biological system completed by a spent media metabolome analysis. Gastrulating 9.5‐day‐old rat embryos‐proper were cultivated in Eagle's minimal essential medium (MEM) with 50% rat serum (controls) or with the addition of 2 mm VPA. Spent media metabolomes were analyzed by FTIR. Compared to controls, VPA acetylated histones; significantly diminished overall teratoma growth, impaired survival, increased the apoptotic index, and decreased proliferation index and incidence of differentiated tissues (e.g., neural tissue). Control teratomas continued to grow and differentiate for 14 days in isotransplants in vivo, but in vitro VPA‐treated teratomas resorbed. Principal component analysis of FTIR results showed that spent media metabolomes formed well‐separated clusters reflecting the treatment and day of cultivation. In metabolomes of VPA‐treated teratomas, we found elevation of previously described histone acetylation biomarkers [amide I α‐helix and A(CH3)/A(CH2)]) with apoptotic biomarkers within the amide I region for β‐sheets, and unordered and CH 2 vibrations of lipids. VPA may be proposed for therapy of the undifferentiated component of teratoma tumors and this biological system completed by metabolome analysis, for a faster dual screening of antitumor/embryotoxic agents.

Tissue inhibitor of metalloproteinase proteins inhibit teratoma growth in mice transplanted with pluripotent stem cells.

Pluripotent stem cells (PSCs) can serve as an unlimited cell source for transplantation therapies for treating various devastating diseases, such as cardiovascular diseases, diabetes, and Parkinson's disease. However, PSC transplantation has some associated risks, including teratoma formation from the remaining undifferentiated PSCs. Thus, for successful clinical application, it is essential to ablate the proliferative PSCs before or after transplantation. In this study, neural stem cell-derived conditioned medium (NSC-CM) inhibited the proliferation of PSCs and PSC-derived neural precursor (NP) cells without influencing the potential of PSC-NP cells to differentiate into neurons in vitro and prevented teratoma growth in vivo. Moreover, we found that the NSC-CM remarkably decreased the expression levels of Oct4 and cyclin D1 that Oct4 directly binds to and increased the cleaved-caspase 3-positive cell death through the DNA damage response in PSCs and PSC-NPs. Interestingly, we found that NSCs distinctly secreted the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 proteins. These proteins suppressed not only the proliferation of PSCs in cell culture but also teratoma growth in mice transplanted with PSCs through inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 activity. Taken together, these results suggest that the TIMP proteins may improve the efficacy and safety of the PSC-based transplantation therapy.

Silencing of the transcription factors Oct4, Sox2, Klf4, c-Myc or Nanog has different effect on teratoma growth

Induced pluripotent stem cells (iPSC) have a great potential, but their clinical application depends on finding strategies to abolish their tumorigenic potential. The use of Oct4, Sox2, Klf4, c-Myc and Nanog to generate iPSC demonstrated the already known importance of these genes to maintain stemness. Therefore, the presence of these genes is responsible for iPSC-derived teratomas. Similar to iPSC, P19 teratocarcinoma cell line also has characteristics of embryonic carcinoma cells and the ability to differentiate into many cell types. We separately silenced the transcription factors Oct4, Sox2, Klf4, c-Myc and Nanog in P19 cells and measured the impact of this silencing in vivo. All silenced cells generated tumors when injected in immunosuppressed mice, but silencing of Oct4, Sox2 and Klf4 generated mainly teratomas with mesoderm tissue. Our results suggest that downregulation of these transcription factors is not enough to avoid the formation of teratomas, but their silencing affect their differentiation potential.

The human somatostatin receptor type 2 as an imaging and suicide reporter gene for pluripotent stem cell-derived therapy of myocardial infarction.

Rationale: Pluripotent stem cells (PSCs) are being investigated as a cell source for regenerative medicine since they provide an infinitive pool of cells that are able to differentiate towards every cell type of the body. One possible therapeutic application involves the use of these cells to treat myocardial infarction (MI), a condition where billions of cardiomyocytes (CMs) are lost. Although several protocols have been developed to differentiate PSCs towards CMs, none of these provide a completely pure population, thereby still posing a risk for neoplastic teratoma formation. Therefore, we developed a strategy to (i) monitor cell behavior noninvasively via site-specific integration of firefly luciferase (Fluc) and the human positron emission tomography (PET) imaging reporter genes, sodium iodide symporter (hNIS) and somatostatin receptor type 2 (hSSTr2), and (ii) perform hSSTr2-mediated suicide gene therapy via the clinically used radiopharmacon 177Lu-DOTATATE.Methods: Human embryonic stem cells (ESCs) were gene-edited via zinc finger nucleases to express Fluc and either hNIS or hSSTr2 in the safe harbor locus, adeno-associated virus integration site 1. Firstly, these cells were exposed to 4.8 MBq 177Lu-DOTATATE in vitro and cell survival was monitored via bioluminescence imaging (BLI). Afterwards, hNIS+ and hSSTr2+ ESCs were transplanted subcutaneously and teratomas were allowed to form. At day 59, baseline 124I and 68Ga-DOTATATE PET and BLI scans were performed. The day after, animals received either saline or 55 MBq 177Lu-DOTATATE. Weekly BLI scans were performed, accompanied by 124I and 68Ga-DOTATATE PET scans at days 87 and 88, respectively. Finally, hSSTr2+ ESCs were differentiated towards CMs and transplanted intramyocardially in the border zone of an infarct that was induced by left anterior descending coronary artery ligation. After transplantation, the animals were monitored via BLI and PET, while global cardiac function was evaluated using cardiac magnetic resonance imaging.Results: Teratoma growth of both hNIS+ and hSSTr2+ ESCs could be followed noninvasively over time by both PET and BLI. After 177Lu-DOTATATE administration, successful cell killing of the hSSTr2+ ESCs was achieved both in vitro and in vivo, indicated by reductions in total tracer lesion uptake, BLI signal and teratoma volume. As undifferentiated hSSTr2+ ESCs are not therapeutically relevant, they were differentiated towards CMs and injected in immune-deficient mice with a MI. Long-term cell survival could be monitored without uncontrolled cell proliferation. However, no improvement in the left ventricular ejection fraction was observed.Conclusion: We developed isogenic hSSTr2-expressing ESCs that allow noninvasive cell monitoring in the context of PSC-derived regenerative therapy. Furthermore, we are the first to use the hSSTr2 not only as an imaging reporter gene, but also as a suicide mechanism for radionuclide therapy in the setting of PSC-derived cell treatment.

Efficient Killing of Murine Pluripotent Stem Cells by Natural Killer (NK) Cells Requires Activation by Cytokines and Partly Depends on the Activating NK Receptor NKG2D.

Natural killer (NK) cells play an important role as cytotoxic effector cells, which scan the organism for infected or tumorigenic cells. Conflicting data have been published whether NK cells can also kill allogeneic or even autologous pluripotent stem cells (PSCs) and which receptors are involved. A clarification of this question is relevant since an activity of NK cells against PSCs could reduce the risk of teratoma growth after transplantation of PSC-derived grafts. Therefore, the hypothesis has been tested that the activity of NK cells against PSCs depends on cytokine activation and specifically on the activating NK receptor NKG2D. It is shown that a subcutaneous injection of autologous iPSCs failed to activate NK cells against these iPSCs and can give rise to teratomas. In agreement with this result, several PSC lines, including two iPSC, two embryonic stem cell (ESC), and two so-called multipotent adult germline stem cell (maGSC) lines, were largely resistant against resting NK cells although differences in killing were found at low level. All PSC lines were killed by interleukin (IL)-2-activated NK cells, and maGSCs were better killed than the other PSC types. The PSCs expressed ligands of the activating NK receptor NKG2D and NKG2D-deficient NK cells from Klrk1−/− mice were impaired in their cytotoxic activity against PSCs. The low-cytotoxic activity of resting NK cells was almost completely dependent on NKG2D. The cytotoxic activity of IL-2-activated NKG2D-deficient NK cells against PSCs was reduced, indicating that also other activating receptors on cytokine-activated NK cells must be engaged by ligands on PSCs. Thus, NKG2D is an important activating receptor involved in killing of murine PSCs. However, NK cells need to be activated by cytokines before they efficiently target PSCs and then also other NK receptors become relevant. These features of NK cells might be relevant for transplantation of PSC-derived grafts since NK cells have the capability to kill undifferentiated cells, which might be present in grafts in trace amounts.

Brief Report: External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1×103 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. Stem Cells 2017;35:1994-2000.

KBP-1 Supports Acute Myeloid Leukemia Development

Novel targets are needed to develop effective therapeutic approaches to treat acute myeloid leukemia (AML). We have developed a systematic strategy to identify factors important for leukemia development. We first use clinical databases to identify plasma membrane proteins that have correlations with the clinical outcomes of leukemia patients. We then validate the functions of candidate proteins in leukemia models and compare these functions to those in normal cells. The signaling pathways identified provide candidate targets for development of therapeutic approaches. Using this approach, we previously identified several ITIM-containing receptors that support AML development.Here we performed an in silico analysis of the relationship between gene expression and the overall survival of AML patients using data from three independent databases: the TCGA AML database (https://tcga-data.nci.nih.gov/tcga/; n = 187), the GSE6891 database (n = 520), and the GSE10358 database (n = 91). Expression of genes encoding several factors, including IL2RA, GPR56, ACDY7, and kappa-binding protein-1 (KBP-1), inversely correlated with the overall survival of AML patients. We focused on the potential function of KBP-1 in AML development in this study.KBP-1 is a transcriptional regulator that was known to inhibit NF-kB signaling and enhance TGF-beta signaling. Previous studies indicated that KBP-1 inhibits teratoma growth. We detected significantly higher kbp-1 mRNA levels in cells from human AML cells than other leukemia cells. To study the potential function of KBP-1 in human leukemia, we inhibited the expression of KBP-1 by introducing lentivirus-encoded shRNAs into MV4-11 and THP-1 cells. The KBP-1 deficiency resulted in significantly decreased in vitro growth of these leukemia cells over time. To determine the underlying mechanism by which KBP-1 supports the growth of leukemia cells, we compared the cell cycle status, migration, and apoptosis of AML cells treated with these shRNAs or scrambled control shRNA. KBP-1-deficient cells had significantly increased levels of apoptosis compared to cells treated with the control shRNA (for example, 68% early apoptosis in KBP-1 knockdown MV4-11 cells vs 15% early apoptosis in control MV4-11 cells at 3 days after shRNA infection). These results indicate that KBP-1 supports leukemia cell growth by suppressing apoptosis.We further studied the function of KBP-1 in AML development using the KBP-1 knockout mice and retrovirus transplantation mouse models. Consistent with the results of KBP-1 knockdown in human leukemia cells, the knockout of KBP-1 in MLL-AF9 AML cells dramatically delayed AML development in mice, as determined by survival, flow cytometry, immunohistochemistry, and colony forming analyses. Serial transplantation of wild-type and KBP-1 knockout AML cells indicates that KBP-1 deficiency impaired the self-renewal of AML stem cells. We are working on elucidating the underlying mechanism by which KBP-1 supports the activity of AML stem cells. Together, this is the first demonstration of KBP-1 function in hematopoietic maligancies. KBP-1 is highly expressed by AML cells and its expression correlates with AML development. KBP-1 supports the survival of human AML cells and the activity of AML stem cells. The tumor-supportive role of KBP-1 in AML is different from its tumor-suppressive function in teratoma. DisclosuresNo relevant conflicts of interest to declare.

Teratoma formation: a tool for monitoring pluripotency in stem cell research.

This unit describes protocols for evaluating the pluripotency of embryonic and induced pluripotent stem cells using a teratoma formation assay. Cells are prepared for injection and transplanted into immunodeficient mice at the gastrocnemius muscle, a site well suited for teratoma growth and surgical access. Teratomas that form from the cell transplants are explanted, fixed in paraformaldehyde, and embedded in paraffin. These preserved samples are sectioned, stained, and analyzed. Pluripotency of a cell line is confirmed by whether the teratoma contains tissues derived from each of the embryonic germ layers: endoderm, mesoderm, and ectoderm. Alternatively, explanted and fixed teratomas can be cryopreserved for immunohistochemistry, which allows for more detailed identification of specific tissue types present in the samples.

Deleting maternal Gtl2 leads to growth enhancement and decreased expression of stem cell markers in teratoma.

The distal region of mouse chromosome 12 harbors the Dlk1–Dio3 domain, is essential for normal development and encodes maternally expressed noncoding RNAs (ncRNAs), including Gtl2 as well as paternally expressed proteins.Gtl2 works as a tumor suppressor in several types of human cancer cell lines; however, whether this reflects its function in vivo is unknown. Deleting Gtl2 from the maternal allele (Gtl2(–/+)) results in loss of expression of Gtl2 and decreased expression of downstream ncRNAs, including many miRNAs. To determine the role of ncRNAs in tumorigenesis, we induced teratomas by engrafting E6.5 embryos (wildtype or Gtl2(–/+)) under the kidney capsule of scid mice. Some teratomas derived from the Gtl2(–/+) embryos exhibited hypertrophic growth, suggesting that ncRNAs, including Gtl2, may act as tumor suppressors in vivo. Microarray analysis of miRNAs expressed by Gtl2(–/+) teratomas revealed decreased expression of 28 miRNAs encoded by the Dlk1–Dio3 domain, low expression of embryonic stem cell-specific miRNAs and dysregulation of miRNAs involved in tumorigenesis. This study suggests that downregulation of ncRNAs in the Dlk1-Dio3 domain leads to enhanced teratoma growth and repression of stem cell markers.

Non-viral reprogramming of fibroblasts into induced pluripotent stem cells by Sleeping Beauty and piggyBac transposons

The generation of induced pluripotent stem (iPS) cells represents a promising approach for innovative cell therapies. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of tumorigenicity. Transposition of reprogramming cassettes represents a recent alternative to viral approaches. Since binary transposons can be produced as common plasmids they provide a safe and cost-efficient alternative to viral delivery methods. Here, we compared the efficiency of two different transposon systems, Sleeping Beauty (SB) and piggyBac (PB), for the generation of murine iPS. Murine fibroblasts derived from an inbred BL/6 mouse line carrying a pluripotency reporter, Oct4-EGFP, and fibroblasts derived from outbred NMRI mice were employed for reprogramming. Both transposon systems resulted in the successful isolation of murine iPS cell lines. The reduction of the core reprogramming factors to omit the proto-oncogene c-Myc was compatible with iPS cell line derivation, albeit with reduced reprogramming efficiencies. The transposon-derived iPS cells featured typical hallmarks of pluripotency, including teratoma growth in immunodeficient mice. Thus SB and PB transposons represent a promising non-viral approach for iPS cell derivation.

Pleiotropic Effects of the Trichloroethylene-Associated P81S VHL Mutation on Metabolism, Apoptosis, and ATM-Mediated DNA Damage Response

BackgroundThe risk relevance of the P81S von Hippel-Lindau (VHL) gene hotspot mutation identified in clear cell renal cell carcinoma from individuals exposed occupationally to trichloroethylene (TCE) is not known. VHL mutations in hereditary VHL syndrome strongly correlate with phenotypic associations, but specific sporadic mutations in VHL that uniquely alter its protein function may provide a selective growth advantage for somatic cells harboring these mutations.MethodsVHL deficient (Vhl -/-) mouse embryonic stem cells were generated that stably express wild-type, P81S, or R167Q human VHL protein. Under hypoxic conditions, cell lines were examined for hypoxia-inducible transcription factor family (HIF) stabilization and E3-ubiquitin ligase complex interactions. In vivo, teratomas were examined for tumor size, proliferation, apoptosis, and immunohistochemistry and subjected to gene expression analysis. Wild-type, R167Q, and P81S VHL-expressing teratomas were also exposed to 5 Gy ionizing radiation to quantify apoptotic response. Proliferation and apoptosis and teratoma growth were analyzed by either Student t test or analysis of variance with Bonferroni correction. All statistical tests were two-sided.ResultsThe P81S VHL mutation produces deregulation of HIF factors in cell culture but exhibits a growth advantage in the tumor microenvironment, in part because of suppression of apoptosis (P81S mean = 0.9%, 95% confidence interval = 0.6 to 1.2%; WT mean = 7.6%; 95% confidence interval = 6.4 to 8.8%; P < .001) coupled with sustained proliferation. Transcriptional analysis of P81S teratomas revealed the induction of metabolic pathways, antiapoptotic genes, and global suppression of key DNA damage response genes not observed in VHL wild-type or R167Q mutants. In vivo irradiation exposure showed that P81S mutant is resistant to ionizing radiation–induced apoptosis.ConclusionsThe TCE-associated P81S VHL mutation can initiate a unique adaptive response required for selective tumor growth through pleiotropic effects on metabolic diversification, apoptosis suppression, and alteration of the DNA damage response.

A benign psoas teratoma of the non‐pelvic retroperitoneal space: A case report

This case report is of a benign retroperitoneal teratoma which was adherent to the psoas muscle of a 53-year-old female cadaver with a history of metastatic bladder cancer as a cause of death. Teratomas possess derivatives of all three germ cell layers and are usually seen either in children or in the lower pelvic region of adults. The finding of this mass deep to the inferior pole of the kidney is quite unusual. Few studies mention a possible association between metastatic cancer and teratoma growth in extrapelvic locations. The following report is a detailed account of this unusual finding and a summary of the literature on growths of this kind.

MIF Produced by Bone Marrow–Derived Macrophages Contributes to Teratoma Progression after Embryonic Stem Cell Transplantation

Although stem cell therapy holds promise as a potential treatment in a number of diseases, the tumorigenicity of embryonic stem cells (ESC) and induced pluripotent stem cells remains a major obstacle. In vitro predifferentiation of ESCs can help prevent the risk of teratoma formation, yet proliferating neural progenitors can generate tumors, especially in the presence of immunosuppressive therapy. In this study, we investigated the effects of the microenvironment on stem cell growth and teratoma development using undifferentiated ESCs. Syngeneic ESC transplantation triggered an inflammatory response that involved the recruitment of bone marrow (BM)-derived macrophages. These macrophages differentiated into an M2 or angiogenic phenotype that expressed multiple angiogenic growth factors and proteinases, such as macrophage migration inhibitory factor (MIF), VEGF, and matrix metalloproteinase 9, creating a microenvironment that supported the initiation of teratoma development. Genetic deletion of MIF from the host but not from ESCs specifically reduced angiogenesis and teratoma growth, and MIF inhibition effectively reduced teratoma development after ESC transplantation. Together, our findings show that syngeneic ESC transplantation provokes an inflammatory response that involves the rapid recruitment and activation of BM-derived macrophages, which may be a crucial driving force in the initiation and progression of teratomas.

Proteomic analysis of mesenchymal stem‐like cells derived from ovarian teratoma: Potential role of glutathione S‐transferase M2 in ovarian teratoma

Ovarian teratoma is a dermoid cyst in the ovary that contains mature tissues such as hair, teeth, bone, thyroid, etc. To understand the molecular mechanisms of ovarian teratoma growth, a comparative proteomic analysis was undertaken using mesenchymal stem cell-like cells (MSCLCs) isolated from normal human ovarian or teratoma tissues. Both normal ovarian and teratoma MSCLCs expressed stem cell markers OCT4 and NANOG, and were negatively staining with the senescence-associated (SA) β-galactosidase. Furthermore, teratoma MSCLCs had higher proliferation and colony formation rates, with more angiogenic property than that of normal MSCLCs. Proteomic study revealed that 17 proteins had the expression changes over eightfold in ovarian teratoma MSCLCs compared with normal control. Interestingly, among them, GSTM2 was strongly expressed in teratoma MSCLCs. Moreover, overexpressed GSTM2 in the teratoma was associated with downregulation of p38 MAPK and activation of AKT and survivin. Taken together, these findings suggest that that ovarian teratoma MSCLCs have a higher potency for proliferation and angiogenesis and GSTM2 appears to be involved in the regulation of other survival genes.

Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic, and xenogeneic natural killer cells

Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.

Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

BackgroundMultipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs).ResultsWe have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide.ConclusionPluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells.ReviewersThis article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter.

Establishment of Endoderm Progenitors by SOX Transcription Factor Expression in Human Embryonic Stem Cells

In this study, we explore endoderm cell fate regulation through the expression of lineage-determining transcription factors. We demonstrate that stable endoderm progenitors can be established from human ES cells by constitutive expression of SOX7 or SOX17, producing extraembryonic endoderm and definitive endoderm progenitors, respectively. In teratoma assays and growth factor-mediated differentiation, SOX7 cells appear restricted to the extraembryonic endoderm, and SOX17 cells demonstrate a mesendodermal phenotype in teratomas and the ability to undergo endoderm maturation in vitro in the absence of cytokine-mediated endoderm induction. These endoderm progenitor cells maintain a stable phenotype through many passages in culture, thereby providing new tools to explore the pathways of endoderm differentiation.

Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines

For clinical grade human embryonic stem cell (hESC) lines, a robust derivation system without any substances having animal origin would be required. We have gradually improved our hESC derivations. Human skin fibroblasts were used as feeder cells in derivation of all our 25 permanent fully characterized hESC lines. In the first four derivations, fetal calf serum was used as a supplement in the medium, thereafter, serum replacement medium was used. Immunosurgery generally used for isolation of the inner cell mass (ICM) still involves animal serum and complement. We developed a practical mechanical isolation method for the ICM. Two flexible metal needles with sharpened tips, 0.125 mm in diameter, were used to open the zona pellucida and extract the ICM under a stereomicroscope. Immunohistochemical and karyotype characterization of the new hESC lines was carried out, and pluripotency was tested in vitro (immunocytochemistry and RT-PCR) and in vivo (teratoma growth). Five hESC lines were obtained from 19 supernumerary blastocysts collected in 2005-2006 (26%), whereas in similar conditions, we obtained 16 lines from 100 blastocysts (16%) using immunosurgery in 2003-2005. The new lines had a normal karyotype and tissues originating from the three embryonic germ cell layers were present. Mechanical isolation of the ICM proved to be an effective way to derive new hESC lines. The technique is fast, does not require any extra investment and the xeno-components of immunosurgery could be avoided.