Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures.

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.

Tissue-specific and transcription-dependent mechanisms regulate primary microRNA processing efficiency of the human chromosome 19 MicroRNA cluster.

One of the longest human microRNA (miRNA) clusters is located on chromosome 19 (C19MC), containing 46 miRNA genes, which were considered to be expressed simultaneously and at similar levels from a common long noncoding transcript. Investigating the two tissue types where C19MC is exclusively expressed, we could show that there is a tissue-specific and chromosomal position-dependent decrease in mature miRNA levels towards the 3' end of the cluster in embryonic stem cells but not in placenta. Although C19MC transcription level is significantly lower in stem cells, this gradual decrease is not present at the primary miRNA levels, indicating that a difference in posttranscriptional processing could explain this observation. By depleting Drosha, the nuclease component of the Microprocessor complex, we could further enhance the positional decrease in stem cells, demonstrating that a tissue-specific, local availability of the Microprocessor complex could lie behind the phenomenon. Moreover, we could describe a tissue-specific promoter being exclusively active in placenta, and the epigenetic mark analysis suggested the presence of several putative enhancer sequences in this region. Performing specific chromatin immunoprecipitation followed by quantitative real-time PCR experiments we could show a strong association of Drosha with selected enhancer regions in placenta, but not in embryonic stem cells. These enhancers could provide explanation for a more efficient co-transcriptional recruitment of the Microprocessor, and therefore a more efficient processing of pri-miRNAs throughout the cluster in placenta. Our results point towards a new model where tissue-specific, posttranscriptional 'fine-tuning' can differentiate among miRNAs that are expressed simultaneously from a common precursor.

Functional Comparison of Blood-Derived Human Neural Progenitor Cells.

Induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) are promising tools to model complex neurological or psychiatric diseases, including schizophrenia. Multiple studies have compared patient-derived and healthy control NPCs derived from iPSCs in order to investigate cellular phenotypes of this disease, although the establishment, stabilization, and directed differentiation of iPSC lines are rather expensive and time-demanding. However, interrupted reprogramming by omitting the stabilization of iPSCs may allow for the generation of a plastic stage of the cells and thus provide a shortcut to derive NPSCs directly from tissue samples. Here, we demonstrate a method to generate shortcut NPCs (sNPCs) from blood mononuclear cells and present a detailed comparison of these sNPCs with NPCs obtained from the same blood samples through stable iPSC clones and a subsequent neural differentiation (classical NPCs-cNPCs). Peripheral blood cells were obtained from a schizophrenia patient and his two healthy parents (a case-parent trio), while a further umbilical cord blood sample was obtained from the cord of a healthy new-born. The expression of stage-specific markers in sNPCs and cNPCs were compared both at the protein and RNA levels. We also performed functional tests to investigate Wnt and glutamate signaling and the oxidative stress, as these pathways have been suggested to play important roles in the pathophysiology of schizophrenia. We found similar responses in the two types of NPCs, suggesting that the shortcut procedure provides sNPCs, allowing an efficient screening of disease-related phenotypes.

Functional characterization of the ABCG2 5' non-coding exon variants: Stem cell specificity, translation efficiency and the influence of drug selection.

ABCG2 is a multidrug transporter with wide substrate specificity, and is believed to protect several cell types from various xenobiotics and endobiotics. This "guardian" function is important in numerous cell types and tissue barriers but becomes disadvantageous by being responsible for the multidrug resistance phenotype in certain tumor cells. ABCG2 regulation at the protein level has already been extensively studied, however, regulation at the mRNA level, especially the functional role of the various 5' untranslated exon variants (5' UTRs) has been elusive. In the present work, we describe a comprehensive characterization of four ABCG2 mRNA variants with different exon 1 sequences, investigate drug inducibility, stem cell specificity, mRNA stability, and translation efficiency. Although certain variants (E1B and E1C) are considered as "constitutive" mRNA isoforms, we show that chemotoxic drugs significantly alter the expression pattern of distinct ABCG2 mRNA isoforms. When examining human embryonic stem cell lines, we provide evidence that variant E1A has an expression pattern coupled to undifferentiated stem cell stage, as its transcript level is regulated parallel to mRNAs of Oct4 and Nanog pluripotency marker genes. When characterizing the four exon 1 variants we found no significant differences in terms of mRNA stabilities and half-lives of the isoforms. In contrast, variant E1U showed markedly lower translation efficiency both at the total protein level or regarding the functional presence in the plasma membrane. Taken together, these results indicate that the different 5' UTR variants play an important role in cell type specific regulation and fine tuning of ABCG2 expression.

Characterization and role of SCAI during renal fibrosis and epithelial-to-mesenchymal transition.

During progressive tubulointerstitial fibrosis, renal tubular epithelial cells transform into α-smooth muscle actin (SMA)-expressing myofibroblasts via epithelial-to-mesenchymal transition (EMT). SMA expression is regulated by transforming growth factor (TGF)-ß1 and cell contact disruption, through signaling events targeting the serum response factor-myocardin-related transcription factor (MRTF) complex. MRTFs are important regulators of fibrosis, tumor cell invasion, and metastasis. Consistent with the role of MRTFs in tumor progression, suppressor of cancer cell invasion (SCAI) was recently identified as a negative regulator of MRTF. Herein, we studied the role of SCAI in a fibrotic EMT model established on LLC-PK1 cells. SCAI overexpression prevented SMA promoter activation induced by TGF-ß1. When co-expressed, it inhibited the stimulatory effects of MRTF-A, MRTF-B or the constitutive active forms of RhoA, Rac1, or Cdc42 on the SMA promoter. SCAI interfered with TGF-ß1-induced SMA, connective tissue growth factor, and calponin protein expression; it rescued TGF-ß1-induced E-cadherin down-regulation. IHC studies on human kidneys showed that SCAI expression is reduced during fibrosis. Kidneys of diabetic rats and mice with unilateral ureteral obstruction depicted significant loss of SCAI expression. In parallel with the decrease of SCAI protein expression, diabetic rat and mouse kidneys with unilateral ureteral obstruction showed SMA expression, as evidenced by using Western blot analysis. Finally, TGF-ß1 treatment of LLC-PK1 cells attenuated SCAI protein expression. These data suggest that SCAI is a novel transcriptional cofactor that regulates EMT and renal fibrosis.

Dynamic ABCG2 expression in human embryonic stem cells provides the basis for stress response.

ABCG2 is a plasma membrane multidrug transporter with an established role in the cancer drug-resistance phenotype. This protein is expressed in a variety of tissues, including several types of stem cell. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines, with a heterogeneous expression pattern. In this study we examined this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between expression of pluripotency markers in ABCG2-positive and negative hESCs; however, ABCG2-expressing cells had a higher growth rate after cell separation. We found that some harmful conditions (physical stress, drugs, and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused rapid internalization of ABCG2, indicating that some environmental factors may induce removal of this transporter from the plasma membrane. On the basis of these results we suggest that a dynamic balance of ABCG2 expression at the population level has the advantage of enabling prompt response to changes in the cellular environment. Such actively maintained heterogeneity might be of evolutionary benefit in protecting special cell types, including pluripotent stem cells.

Generation of Human Neural Progenitors from Blood Samples by Interrupted Reprogramming.

Human neuronal cell cultures are essential tools for biological and preclinical studies of our nervous system. Since we have very limited access to primary human neural samples, derivation of proliferative neural progenitor cells (NPCs) from cells harvested by minimally invasive sampling is a key issue. Here we describe a "shortcut" method to establish proliferative NPC cultures directly from peripheral blood mononuclear cells (PBMCs) via interrupted reprogramming. In addition, we provide procedures to characterize the NPC stage.

Comparison of Different Clinical Chemotherapeutical Agents' Toxicity and Cell Response on Mesenchymal Stem Cells and Cancer Cells.

Mesenchymal stem cells (MSCs) or fibroblasts are one of the most abundant cell types in the tumor microenvironment (TME) exerting various anti- and pro-apoptotic effects during tumorigenesis, invasion, and drug treatment. Despite the recently discovered importance of MSCs in tumor progression and therapy, the response of these cells to chemotherapeutics compared to cancer cells is rarely investigated. A widely accepted view is that these naive MSCs have higher drug tolerance than cancer cells due to a significantly lower proliferation rate. Here, we examine the differences and similarities in the sensitivity of MSCs and cancer cells to nine diverse chemotherapy agents and show that, although MSCs have a slower cell cycle, these cells are still sensitive to various drugs. Surprisingly, MSCs showed similar sensitivity to a panel of compounds, however, suffered fewer DNA double-stranded breaks, did not enter into a senescent state, and was virtually incapable of apoptosis. Our results suggest that MSCs and cancer cells have different cell fates after drug treatment, and this could influence therapy outcome. These findings could help design drug combinations targeting both MSCs and cancer cells in the TME.

Generation of multiple iPSC clones from a male schizophrenia patient carrying de novo mutations in genes KHSRP, LRRC7, and KIR2DL1, and his parents.

Here we describe the generation of induced pluripotent stem cell lines from each member - male proband, mother, father - of a schizophrenia case-parent trio that participated in an exome sequencing study, and 3 de novo mutations were identified in the proband. Peripheral blood mononuclear cells were obtained from all three individuals and reprogrammed using Sendai virus particles carrying the Yamanaka transgenes. These 3 iPSC lines (iPSC-SZ-HU-MO 1, iPSC-SZ-HU-FA 1, and iPSC-SZ-HU-PROB 1) represent a resource for examining the functional significance of the identified de novo mutations in the molecular pathophysiology of schizophrenia.

Applying a "double-feature" promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery.

Human embryonic stem (HuES) cells represent a new potential tool for cell-therapy and gene-therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines, we have generated stable, enhanced green fluorescent protein (EGFP)-expressing clones using a transposon-based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, elongation factor 1alpha, or phosphoglycerate kinase), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This "double-feature" promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter-driven EGFP transcription and expression of cardiomyocyte-specific genes. Our experiments indicate an efficient applicability of transposon-based gene delivery into HuES cells and provide a novel approach to identify differentiated tissues by exploiting a nontypical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods.

Cdc42 regulates myocardin-related transcription factor nuclear shuttling and alpha-smooth muscle actin promoter activity during renal tubular epithelial-mesenchymal transition.

BACKGROUND/AIMS: Epithelial-mesenchymal transition of tubular cells into alpha-smooth muscle actin (SMA)-expressing myofibroblasts is a central mechanism in tubulointerstitial fibrosis. Previously, a 'two-hit' model was proposed for epithelial-mesenchymal transition wherein an initial injury of the intercellular contacts and TGF-beta(1) are both required for SMA protein expression in LLC-PK1 cells. The Rho-Rho kinase-myosin light chain-myocardin-related transcription factor (MRTF)-serum response factor (SRF) pathway and Rac1, p21-activated kinase (PAK) and p38 were described as important regulators of MRTF localization and SMA expression. Cdc42 is another small G protein situated upstream of PAK and p38, and is activated upon cell contact disassembly. Here, we investigated its potential role in the regulation of MRTF nuclear shuttling and in the regulation of the SMA promoter. RESULTS: Transfection of a constitutive active (CA) Cdc42 construct alone induced the activation of the SMA promoter. The dominant negative (DN) Cdc42 construct prevented the activation of the promoter induced by cell contact disassembly, and reduced the combined effect of cell contact disruption and TGF-beta(1). SRF showed a marked nuclear accumulation in CA Cdc42-transfected cells. Cdc42 induced the nuclear translocation of MRTF, while DN Cdc42 inhibited its nuclear translocation induced by cell contact disassembly. Blocking PAK, MRTF and p38 by the corresponding DN constructs blunted the effects of CA Cdc42 on the SMA promoter. CONCLUSION: Cdc42 is involved in the regulation of SMA promoter activation through PAK, p38, MRTF and SRF. Cdc42 may be an important regulator of MRTF cellular localization.

The importance of transporters and cell polarization for the evaluation of human stem cell-derived hepatic cells.

One of the most promising applications of human pluripotent stem cells is their utilization for human-based pharmacological models. Despite the fact that membrane transporters expressed in the liver play pivotal role in various hepatic functions, thus far only little attention was devoted to the membrane transporter composition of the stem cell-derived liver models. In the present work, we have differentiated HUES9, a human embryonic stem cell line, toward the hepatic lineage, and monitored the expression levels of numerous differentiation marker and liver transporter genes with special focus on ABC transporters. In addition, the effect of bile acid treatment and polarizing culturing conditions on hepatic maturation has been assessed. We found that most transporter genes crucial for hepatic functions are markedly induced during hepatic differentiation; however, as regards the transporter composition the end-stage cells still exhibited dual, hepatocyte and cholangiocyte character. Although the bile acid treatment and sandwich culturing only slightly influenced the gene expressions, the stimulated cell polarization resulted in formation of bile canaliculi and proper localization of transporters. Our results point to the importance of membrane transporters in human stem cell-derived hepatic models and demonstrate the relevance of cell polarization in generation of applicable cellular models with correctly localized transporters. On the basis of our observations we suggest that conventional criteria for the evaluation of the quality of stem cell-derived hepatocyte-like cells ought to be augmented with additional elements, such as polarized and functional expression of hepatic transporters.

Establishing a human embryonic stem cell clone with a heterozygous mutation in the DGCR8 gene.

DiGeorge Syndrome (DGS) Critical Region 8 (DGCR8) is a primary candidate gene in they DGS. The DGCR8 microprocessor complex subunit is an essential cofactor in the canonical miRNA biogenesis which is involved in diverse cellular functions such as cell fate decisions, apoptosis and different signaling pathways. However, the role of DGCR8 in these processes or development of DGS is not fully understood. Here we present a heterozygous DGCR8 mutant human embryonic stem cell line (HuES9DGCR8+/-) created by the CRISPR/Cas9 system. The generated HuES9DGCR8+/- cells maintain normal karyotype, morphology, pluripotency and differentiation capacity into all three germ layers.

Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell-based in vitro disease modeling: convergence of schizophrenia- and autism-related cellular phenotypes.

BACKGROUND: De novo mutations (DNMs) have been implicated in the etiology of schizophrenia (SZ), a chronic debilitating psychiatric disorder characterized by hallucinations, delusions, cognitive dysfunction, and decreased community functioning. Several DNMs have been identified by examining SZ cases and their unaffected parents; however, in most cases, the biological significance of these mutations remains elusive. To overcome this limitation, we have developed an approach of using induced pluripotent stem cell (iPSC) lines from each member of a SZ case-parent trio, in order to investigate the effects of DNMs in cellular progenies of interest, particularly in dentate gyrus neuronal progenitors. METHODS: We identified a male SZ patient characterized by early disease onset and negative symptoms, who is a carrier of 3 non-synonymous DNMs in genes LRRC7, KHSRP, and KIR2DL1. iPSC lines were generated from his and his parents' peripheral blood mononuclear cells using Sendai virus-based reprogramming and differentiated into neuronal progenitor cells (NPCs) and hippocampal dentate gyrus granule cells. We used RNASeq to explore transcriptomic differences and calcium (Ca2+) imaging, cell proliferation, migration, oxidative stress, and mitochondrial assays to characterize the investigated NPC lines. RESULTS: NPCs derived from the SZ patient exhibited transcriptomic differences related to Wnt signaling, neuronal differentiation, axonal guidance and synaptic function, and decreased Ca2+ reactivity to glutamate. Moreover, we could observe increased cellular proliferation and alterations in mitochondrial quantity and morphology. CONCLUSIONS: The approach of reprograming case-parent trios represents an opportunity for investigating the molecular effects of disease-causing mutations and comparing these in cell lines with reduced variation in genetic background. Our results are indicative of a partial overlap between schizophrenia and autism-related phenotypes in the investigated family. LIMITATIONS: Our study investigated only one family; therefore, the generalizability of findings is limited. We could not derive iPSCs from two other siblings to test for possible genetic effects in the family that are not driven by DNMs. The transcriptomic and functional assays were limited to the NPC stage, although these variables should also be investigated at the mature neuronal stage.

Application of human pluripotent stem cells and pluripotent stem cell-derived cellular models for assessing drug toxicity.

Introduction: Human pluripotent stem cells (hPSCs) are capable of differentiating into all types of cells in the body and so provide suitable toxicology screening systems even for hard-to-obtain human tissues. Since hPSCs can also be generated from differentiated cells and current gene editing technologies allow targeted genome modifications, hPSCs can be applied for drug toxicity screening both in normal and disease-specific models. Targeted hPSC differentiation is still a challenge but cardiac, neuronal or liver cells, and complex cellular models are already available for practical applications. Areas covered: The authors review new gene-editing and cell-biology technologies to generate sensitive toxicity screening systems based on hPSCs. Then the authors present the use of undifferentiated hPSCs for examining embryonic toxicity and discuss drug screening possibilities in hPSC-derived models. The authors focus on the application of human cardiomyocytes, hepatocytes, and neural cultures in toxicity testing, and discuss the recent possibilities for drug screening in a 'body-on-a-chip' model system. Expert opinion: hPSCs and their genetically engineered derivatives provide new possibilities to investigate drug toxicity in human tissues. The key issues in this regard are still the selection and generation of proper model systems, and the interpretation of the results in understanding in vivo drug effects.

Generation of multidrug resistant human tissues by overexpression of the ABCG2 multidrug transporter in embryonic stem cells.

The ABCG2 multidrug transporter provides resistance against various endo- and xenobiotics, and protects the stem cells against toxins and stress conditions. We have shown earlier that a GFP-tagged version of ABCG2 is fully functional and may be used to follow the expression, localization and function of this transporter in living cells. In the present work we have overexpressed GFP-ABCG2, driven by a constitutive (CAG) promoter, in HUES9 human embryonic stem cells. Stem cell clones were generated to express the wild-type and a substrate-mutant (R482G) GFP-ABCG2 variant, by using the Sleeping Beauty transposon system. We found that the stable overexpression of these transgenes did not change the pluripotency and growth properties of the stem cells, nor their differentiation capacity to hepatocytes or cardiomyocytes. ABCG2 overexpression provided increased toxin resistance in the stem cells, and protected the derived cardiomyocytes against doxorubicin toxicity. These studies document the potential of a stable ABCG2 expression for engineering toxin-resistant human pluripotent stem cells and selected stem cell derived tissues.

The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation.

INTRODUCTION: Drug transporters are large transmembrane proteins which catalyse the movement of a wide variety of chemicals, including drugs as well as xeno- and endobiotics through cellular membranes. The major groups of these proteins include the ATP-binding cassette transporters which in eukaryotes work as ATP-fuelled drug 'exporters' and the Solute Carrier transporters, with various transport directions and mechanisms. AREAS COVERED: In this review, we discuss the key ATP-binding cassette and Solute Carrier drug transporters which have been reported to contribute to the function and/or protection of undifferentiated human stem cells and during tissue differentiation. We review the various techniques for studying transporter expression and function in stem cells, and the role of drug transporters in foetal and placental tissues is also discussed. We especially focus on the regulation of transporter expression by factors modulating cell differentiation properties and on the function of the transporters in adjustment to environmental challenges. EXPERT OPINION: The relatively new and as yet unexplored territory of transporters in stem cell biology may rapidly expand and bring important new information regarding the metabolic and epigenetic regulation of 'stemness' and the early differentiation properties. Drug transporters are clearly important protective and regulatory components in stem cells and differentiation.

Efficient generation of human embryonic stem cell-derived cardiac progenitors based on tissue-specific enhanced green fluorescence protein expression.

Cardiac progenitor cells (CPCs) are committed to the cardiac lineage but retain their proliferative capacity before becoming quiescent mature cardiomyocytes (CMs). In medical therapy and research, the use of human pluripotent stem cell-derived CPCs would have several advantages compared with mature CMs, as the progenitors show better engraftment into existing heart tissues, and provide unique potential for cardiovascular developmental as well as for pharmacological studies. Here, we demonstrate that the CAG promoter-driven enhanced green fluorescence protein (EGFP) reporter system enables the identification and isolation of embryonic stem cell-derived CPCs. Tracing of CPCs during differentiation confirmed up-regulation of surface markers, previously described to identify cardiac precursors and early CMs. Isolated CPCs express cardiac lineage-specific transcripts, still have proliferating capacity, and can be re-aggregated into embryoid body-like structures (CAG-EGFP(high) rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFP(high) rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFP(high) rEBs show spontaneous contractions. The method described here provides a powerful tool to generate expandable cultures of pure human CPCs that can be used for exploring early markers of the cardiac lineage, as well as for drug screening or tissue engineering applications.

Excision efficiency is not strongly coupled to transgenic rate: cell type-dependent transposition efficiency of sleeping beauty and piggyBac DNA transposons.

The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, "overproduction inhibition," a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own "in-house" platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2-3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future.