Site-specific integration of adeno-associated virus involves partial duplication of the target locus

A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences.     

Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.     

Generation of genetically modified rats from embryonic stem cells

At present, genetically modified rats have not been generated from ES cells because stable ES cells and a suitable injection method are not available. To monitor the pluripotency of rat ES cells, we generated Oct4-Venus transgenic (Tg) rats via a conventional method, in which Venus is expressed by the Oct4 promoter/enhancer. This monitoring system enabled us to define a significant condition of culture to establish authentic rat ES cells based on a combination of 20% FBS and cell signaling inhibitors for Rho-associated kinase, mitogen-activated protein kinase, TGF-β, and glycogen synthase kinase-3. The rat ES cells expressed ES cell markers such as Oct4, Nanog, Sox2, and Rex1 and retained a normal karyotype. Embryoid bodies and teratomas were also produced from the rat ES cells. All six ES cell lines derived from three different rat strains successfully achieved germline transmission, which strongly depended on the presence of the inhibitors during the injection process. Most importantly, high-quality Tg rats possessing a correct transgene expression pattern were successfully generated via the selection of gene-manipulated ES cell clones through germline transmission. Our rat ES cells should be sufficiently able to receive gene targeting as well as Tg manipulation, thus providing valuable animal models for the study of human diseases.     

Differentiation of mouse embryonic stem cells into hepatocytes in vitro and in vivo

OBJECTIVE: Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages. Cell‐to‐cell contact is important for cell differentiation. Mouse ES cells were cocultured with mouse fetal liver cells and the green fluorescent protein (GFP) positive ES cells were transplanted into rats liver through the portal vein in order to investigate their potential to differentiate into hepatocytes. METHODS: Mouse ES cells were cocultured with the mouse fetal liver cell line, BNL.CL₂. They did not make direct contact; instead the culture media was exchanged freely. After coculture for 48 h, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4 and SEK1 mRNA were assayed by RT‐PCR, and alpha‐fetoprotein by immunohistochemistry. The morphology was investigated by microscopy. After transplantion of the GFP‐positive ES cells, the whole liver was removed from a rat every four days. The liver slices were examined under a fluorescent microscope to detect the GFP‐positive cells. Albumin was detected on the same slices by immunohistochemistry. RESULTS: After coculture with BNL.CL₂ cells, the differentiated ES cells had the same morphology as the BNL.CL₂ cells, and albumin, transthyretin, glucose 6 phosphates and SEK‐1 mRNA were found by RT‐PCR, and alpha‐fetoprotein was detected immuno­histochemically. The transplanted GFP‐positive ES cells were found in the rats’ liver slices by GFP fluorescence, and development of teratomas was not observed. The immunohistochemistry results indicated that the transplanted GFP‐positive ES cells retained an albumin‐producing ability. CONCLUSIONS: Cell‐to‐cell contact is important for the differentiation of ES cells. Mouse embryonic stem cells can differentiate into hepatocytes directly either in vitro or in vivo.     

Verteporfin synergizes the efficacy of anti-PD-1 in cholangiocarcinoma

BackgroundCholangiocarcinoma (CCA) is one of the primary hepatobiliary malignant neoplasms with only 10% of 5-year survival rate. Promising immunotherapy with the blockade of immune checkpoints has no clear benefit in CCA. The inhibition of YAP1 signaling by verteporfin has shown encouraging results by inhibiting cell proliferation and inducing apoptosis. This study aimed to evaluate the potential benefit of the combination of verteporfin and anti-programmed cell death 1 (PD-1) in CCA mouse model.MethodsWe assessed the cytotoxicity of verteporfin in human CCA cell lines in vitro, including both intrahepatic CCA and extrahepatic CCA cells. We examined the in vitro effect of verteporfin on cell proliferation, apoptosis, and stemness. We evaluated the in vivo efficacy of verteporfin, anti-PD-1, and a combination of both in subcutaneous CCA mouse model.ResultsOur study showed that verteporfin reduced tumor cell growth and enhanced apoptosis of human CCA tumor cells in vitro in a dose-dependent fashion. Nevertheless, verteporfin impaired stemness evidenced by reduced spheroid formation and colony formation, decreased numbers of cells with aldehyde dehydrogenase activity and positive cancer stem cell markers (all P < 0.05). The combination of verteporfin and anti-PD-1 reduced tumor burden in CCA subcutaneous SB1 tumor model compared to either agent alone.ConclusionsVerteporfin exhibits antitumor effects in both intrahepatic and extrahepatic CCA cell lines and the combination with anti-PD-1 inhibited tumor growth.     

Successive generations of mice produced from an established culture line of euploid teratocarcinoma cells

The possibility of utilizing mouse teratocarcinoma stem cells as intermediaries for production of new strains of mice with preselected mutant or foreign genes requires that, after propagation in culture (to allow for genetic manipulation and selection), the cells be capable of normalization and orderly development in carrier embryos and, ultimately, of germ-cell formation. Heretofore, no in vitro cell line has fulfilled all these requirements. A karyotypically normal teratocarcinoma culture line was recently established in this laboratory and now has been investigated as a candidate. The line, designated METT-1, is chromosomally female (X/X) and was obtained from the 129 (agouti-colored) inbred strain [Mintz, B. & Cronmiller, C. (1981) Somat Cell Genet 7, 489-505]. The developmental potential of these cells was tested, after prolonged culture and freezing and thawing, by microinjecting them into early (blastocyst stage) embryos of the C57BL/6 (black) strain. Among 312 experimental animals examined at 1 week of age, there were 41 mice (21 females and 20 males) that displayed the coat colors of both strains. This frequency (13%), as well as the extent of the coat areas derived from the cell line, greatly surpasses the contributions observed in all previous experiments, whether with other in vitro teratocarcinoma cell lines or with in vivo transplant lines. The developmental totipotency of METT-1 cells became evident from the presence of substantial amounts of 129-strain cells (bearing an isozyme marker) in all internal tissues of an individual whose coat was largely agouti. The culture-cell lineage also proved to be capable of giving rise to reproductively functional oocytes. Of nine mosaic-coat females testmated to C57BL/6 males, one produced progeny of the diagnostic agouti color in two litters; these heterozygous F₁ offspring in turn transmitted their marker genes to F₂ homozygous segregants. Thus, the METT-1 teratocarcinoma line bridges the gap between in vitro cell propagation and in vivo development and between the soma and the germ line. This creates the option of producing new mouse strains with predetermined genetic changes designed as probes of developmental regulation or as models of human genetic diseases.     

Embryonic stem cell-derived tissues are immunogenic but their inherent immune privilege promotes the induction of tolerance

Although human embryonic stem (ES) cells may one day provide a renewable source of tissues for cell replacement therapy (CRT), histoincompatibility remains a significant barrier to their clinical application. Current estimates suggest that surprisingly few cell lines may be required to facilitate rudimentary tissue matching. Nevertheless, the degree of disparity between donor and recipient that may prove acceptable, and the extent of matching that is therefore required, remain unknown. To address this issue using a mouse model of CRT, we have derived a panel of ES cell lines that differ from CBA/Ca recipients at defined genetic loci. Here, we show that even expression of minor histocompatibility (mH) antigens is sufficient to provoke acute rejection of tissues differentiated from ES cells. Nevertheless, despite their immunogenicity in vivo, transplantation tolerance may be readily established by using minimal host conditioning with nondepleting monoclonal antibodies specific for the T cell coreceptors, CD4 and CD8. This propensity for tolerance could be attributed to the paucity of professional antigen-presenting cells and the expression of transforming growth factor (TGF)-β₂. Together, these factors contribute to a state of acquired immune privilege that favors the polarization of infiltrating T cells toward a regulatory phenotype. Although the natural privileged status of ES cell-derived tissues is, therefore, insufficient to overcome even mH barriers, our findings suggest it may be harnessed effectively for the induction of dominant tolerance with minimal therapeutic intervention.     

Impact of oncogenes in tumor angiogenesis: Mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells

Targeted disruption of the single mutant K-ras allele in two human colorectal carcinoma cell lines (DLD-1 and HCT-116) leads to loss of tumorigenic competence in nude mice with retention of ability to grow indefinitely in monolayer culture. Because expression of the mutant K-ras oncogene in these cell lines is associated with marked up-regulation of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), we sought to determine whether this potent angiogenesis inducer plays a role in K-ras-dependent tumorigenic competence. Transfection of a VEGF₁₂₁ antisense expression vector into DLD-1 and HCT-116 cells resulted in suppression of VEGF/VPF production by a factor of 3- to 4-fold. The VEGF/VPF-deficient sublines, unlike the parental population or vector controls, were profoundly suppressed in their ability to form tumors in nude mice for as long as 6 months after cell injection. In contrast, in vitro growth of these sublines was unaffected, thus demonstrating the critical importance of VEGF/VPF as an angiogenic factor for HCT-116 and DLD-1 cells. Transfection of a full-length VEGF₁₂₁ cDNA into two nontumorigenic mutant K-ras knockout sublines resulted in a weak but detectable restoration of tumorigenic ability in vivo in a subset of the transfectants, with no consistent change in growth properties in vitro. The findings indicate that mutant ras-oncogene-dependent VEGF/VPF expression is necessary, but not sufficient, for progressive tumor growth in vivo and highlight the relative contribution of oncogenes, such as mutant K-ras, to the process of tumor angiogenesis.     

Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.     

Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells

During differentiation in vitro, embryonic stem (ES) cells generate progenitors for most hemato-lymphoid lineages. We studied the developmental potential of two ES cell subpopulations that share the fetal stem cell antigen AA4.1 but differ in expression of the lymphoid marker B220 (CD45R). Upon transfer into lymphoid deficient mice, the B220⁺ population generated a single transient wave of IgM⁺ IgD⁺ B cells but failed to generate T cells. In contrast, transfer of the B220⁻ fraction achieved long-term repopulation of both T and B lymphoid compartments and restored humoral and cell-mediated immune reactions in the recipients. To assess the hemato-lymphopoietic potential of ES cell subsets in comparison to their physiological counterparts, cotransplantation experiments with phenotypically homologous subsets of fetal liver cells were performed, revealing a more potent developmental capacity of the latter. The results suggest that multipotential and lineage-committed lymphoid precursors are generated during in vitro differentiation of ES cells and that both subsets can undergo complete final maturation in vivo.     

Characterization and culture of human embryonic stem cells

Human embryonic stem (ES) cells are cultured cell lines derived from the inner cell mass of the blastocyst that can be grown indefinitely in their undifferentiated state, yet also are capable of differentiating into all cells of the adult body as well as extraembryonic tissue. Detailed investigation of the properties of embryonal carcinoma cells of both the mouse and human as well as mouse and primate ES cells led to the initial isolation and subsequent culture of human ES cells. The methodologies that were developed to culture and characterize these cell lines have provided a template for the development of human ES cells. The existing data illustrate a number of important differences and similarities between human ES cells and the other cell lines. This review aims to provide a brief historic account of the development of the mammalian pluripotent stem cell field; describe how this led to the isolation, culture, and characterization of human ES cells; and discuss the potential implications of recent advances.     

Developmental and adult phenotyping directly from mutant embryonic stem cells

Tetraploid embryo complementation assay has shown that mouse ES cells alone are capable of supporting embryonic development and adult life of mice. Newly established F(1) hybrid ES cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell-based genetics feasible. Here we report the establishment and characterization of 12 new F(1) hybrid ES cell lines and the use of one of the best (G4) in a gain- and loss-of-function genetic study, where the in vivo phenotypes were assessed directly from ES cell-derived embryos. We found the generation of G4 ES cell-derived animals to be very efficient. Furthermore, even after two consecutive rounds of genetic modifications, the majority of transgenic lines retained the original potential of the parental lines; with 10-40% of chimeras producing ES cell-derived animals/embryos. Using these genetically altered ES cells, this success rate, in most cases, permitted the derivation of a sufficient number of mutants for initial phenotypic analyses only a few weeks after the establishment of the cell lines. Although the experimental design has to take into account a moderate level of uncontrolled damage on ES cell lines, our proof-of-principle experiment provides useful data to assist future designs harnessing the power of this technology to accelerate our understanding of gene function.     

Anti-tumor activity and signaling events triggered by the isothiocyanates, sulforaphane and phenethyl isothiocyanate, in multiple myeloma

Background

Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties.

Design and Methods

We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting.

Results

We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients‘ myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G₂/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/β and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma.

Conclusions

Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.     

Genomewide production of multipurpose alleles for the functional analysis of the mouse genome

A type of retroviral gene trap vectors has been developed that can induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems that can repair and re-induce gene trap mutations when activated in succession. After the gene traps are inserted into the mouse genome, genetic mutations can be produced at a particular time and place in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of post-insertional modifications. Here we have used these directional recombination vectors to assemble the largest library of ES cell lines with conditional mutations in single genes yet assembled, presently totaling 1,000 unique genes. The trapped ES cell lines, which can be ordered from the German Gene Trap Consortium, are freely available to the scientific community.     

Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines

BACKGROUNDColon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers. It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-expressing stem cells, these so-called “tumour-initiating” cells, reminiscent in their properties of the normal intestinal stem cells (ISCs), may explain the apparent heterogeneity of colon cancer cell lines. Also, colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5. Furthermore, in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIMTo investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODSA portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines (Caco-2 and LoVo). These cells were then characterized according to their proliferation capacity, gene expression signatures of ISC markers, and their tumorigenic properties in vivo and in vitro.RESULTSThe data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2, but not from the LoVo, cell lines, in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog (+4 crypt) stem cells. This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny. Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein (EYFP) from this cell line exhibit tumorigenic-like properties in vivo and in vitro. In contrast, cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties. Thus, cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells, but not LoVo cells.CONCLUSIONOur findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines. Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data. We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.     

Regulation of cell proliferation and malignant potential by irisin in endometrial,colon, thyroid and esophageal cancer cell lines

Objective

Irisin is a novel hormone that has been proposed to mediate the beneficial effects of exercise on metabolism, including body weight regulation and insulin resistance. No previous studies have evaluated whether irisin may regulate cell proliferation and malignant potential of obesity-related cancer cell lines.

Materials/Methods

Cell proliferation and malignant potential i.e. cell adhesion and colony formation were studied in vitro using human and mouse obesity-related cancer cell lines i.e. endometrial (KLE and RL95-2), colon (HT29 and MCA38), thyroid (SW579 and BHP7) and esophageal (OE13 and OE33).

Results

We observed that, in contrast to metformin, cell proliferation is not regulated by irisin in a dose-dependent manner in human and mouse obesity-related cancer cell lines. Specifically, physiological (5 to 10 nmol/L) and high physiological/pharmacological (50 to 100 nmol/L) concentrations of irisin had no effect on cell proliferation when compared to control in human and mouse endometrial, colon, thyroid and esophageal cancer cell lines. Also, we observed that, in contrast to metformin, neither physiological nor high physiological/pharmacological concentrations of irisin regulate cell adhesion and/or colony formation in human and mouse endometrial, colon, thyroid and esophageal cancer cell lines.

Conclusions

Our data suggest that irisin, in physiological and high physiological/pharmacological concentrations, has no in vitro effect on cell proliferation and malignant potential of obesity-related cancer cell lines. Future work is needed to determine the regulation of irisin levels and any physiological effects it may have on obesity-related cancers in vivo in animals and humans.     

Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoiesis

HAX1 was identified as the gene responsible for the autosomal recessive type of severe congenital neutropenia. However, the connection between mutations in the HAX1 gene and defective granulopoiesis in this disease has remained unclear, mainly due to the lack of a useful experimental model for this disease. In this study, we generated induced pluripotent stem cell lines from a patient presenting for severe congenital neutropenia with HAX1 gene deficiency, and analyzed their in vitro neutrophil differentiation potential by using a novel serum- and feeder-free directed differentiation culture system. Cytostaining and flow cytometric analyses of myeloid cells differentiated from patient-derived induced pluripotent stem cells showed arrest at the myeloid progenitor stage and apoptotic predisposition, both of which replicated abnormal granulopoiesis. Moreover, lentiviral transduction of the HAX1 cDNA into patient-derived induced pluripotent stem cells reversed disease-related abnormal granulopoiesis. This in vitro neutrophil differentiation system, which uses patient-derived induced pluripotent stem cells for disease investigation, may serve as a novel experimental model and a platform for high-throughput screening of drugs for various congenital neutrophil disorders in the future.     

Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos

The first differentiation event of the mammalian embryo is thought to occur during blastulation and results in two populations of cells, the inner cell mass (ICM) and the trophectoderm. Most embryonic stem (ES) cell lines have been derived from the ICM or a further subset of ICM cells known as the epiblast. There appears to be a limited period of embryonic development during which pluripotent ES cells can be adapted from the cells of the blastocyst to culture. A method is presented here that allows ES cell lines to be isolated from preblastocyst mouse embryos. These lines were derived from 129S2/SvHsd mouse morulae and earlier cleavage stages with high efficiency. The lines expressed genes and antigens characteristic of pluripotent ES cells. XY cell lines remained karyotypically stable through extensive passaging and produced germ-line-competent chimeras upon blastocyst injection. These results suggest that true ES cells can be derived from embryos explanted at any stage of preimplantation development in the mouse. This finding raises the interesting question of whether ES cell lines derived from embryos at different stages of preimplantation development possess the same potential.