Generation of a CAG-EGFP tagged cell line (KSCBi017-A-2) from human induced pluripotent stem cells using CRISPR/Cas9.

Human induced pluripotent stem cells (hiPSCs) have potential use in regerenrative medicine for disease modeling and drug screening studies. The AAVS1 locus has been validated as a stable transgene expression and safe genomic location. Therefore, we inserted the enhanced green fluorescent protein (EGFP) gene into the AAVS1 locus of hiPSCs, using CRISPR/Cas9 genome editing. The results showed that the hiPSCs stably expressed EGFP in pluripotency and differentiated into three germ lineages. Our results strongly indicate that the EGFP-tagged cell line has potential for use in in vivo and in vitro experiments for monitoring cell location and type.

Generation of a human induced pluripotent stem cell line from a patient with Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that is characterized by severe visual impairment in early infancy. We generated a human induced pluripotent stem cell (hiPSC) line, DKHi090-A, from peripheral blood mononuclear cells (PBMCs) of a patient with LCA, by using a Sendai virus-based gene delivery system. We confirmed that DKHi090-A has a nicotinamide mononucleotide adenyltransferase 1 (NMNAT1) mutation and normal karyotype. DKHi090-A line is pluripotent and is capable of multilineage differentiation. This cell line is registered and is available at the National Stem Cell Bank, Korea National Institute of Health.

Generation of human induced pluripotent stem cells from peripheral blood mononuclear cells of a Senior-Loken syndrome patient.

Senior-Loken syndrome (SLS) is a rare disorder primarily associated with kidney and retinal dysfunction. We generated a human induced pluripotency stem cell (hiPSC) line, designated DKHi005-A, from peripheral blood mononuclear cells of a patient with SLS using a Sendai virus reprogramming method. We confirmed that DKHi005-A cells harbor the same mutation as the patient and show a normal karyotype. DKHi005-A also has pluripotency and the capacity for differentiation into the three germ layers. This cell line is registered and available at the National Stem Cell Bank, Korea National Institute of Health.

Generation of a PDX1-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-3, using the CRISPR/Cas9 system.

PDX1 plays a crucial role in the development and maintenance of ß-cells and directly regulates pancreatic ß-cell-specific transcription factors by binding to the insulin gene. Here, we introduced an EGFP reporter into the C-terminus of PDX1 in KSCBi005-A human induced pluripotent stem cells through homologous recombination using CRISPR/Cas9 nuclease. The cells had a normal karyotype, expressed several pluripotency markers, and maintained their differentiation potential. KSCBi005-A-3 cells can be used to monitor PDX1 expression in live cells during ß-cell differentiation; the cell line has been registered at the National Stem Cell Bank, Korea National Institute of Health.

Generation of a NESTIN-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-1, using CRISPR/Cas9 nuclease.

NESTIN, an intermediate filament, is a neuroectodermal marker involved in induced pluripotent stem cell (iPSC) differentiation toward neural lineages. Here, we introduced an EGFP reporter into the C-terminus of NESTIN in KSCBi005-A hiPSCs through homologous recombination using CRISPR/Cas9 nuclease. The successfully edited line was confirmed by sequencing and had a normal karyotype. It expressed EGFP upon induction of neural differentiation and exhibited potential for differentiation into three germ layers. KSCBi005-A-1 cells could be used to monitor the expression of NESTIN in differentiated cell types. This cell line is available at the National Stem Cell Bank, Korea National Institute of Health.

The ID1-CULLIN3 Axis Regulates Intracellular SHH and WNT Signaling in Glioblastoma Stem Cells.

Inhibitor of differentiation 1 (ID1) is highly expressed in glioblastoma stem cells (GSCs). However, the regulatory mechanism responsible for its role in GSCs is poorly understood. Here, we report that ID1 activates GSC proliferation, self-renewal, and tumorigenicity by suppressing CULLIN3 ubiquitin ligase. ID1 induces cell proliferation through increase of CYCLIN E, a target molecule of CULLIN3. ID1 overexpression or CULLIN3 knockdown confers GSC features and tumorigenicity to murine Ink4a/Arf-deficient astrocytes. Proteomics analysis revealed that CULLIN3 interacts with GLI2 and DVL2 and induces their degradation via ubiquitination. Consistent with ID1 knockdown or CULLIN3 overexpression in human GSCs, pharmacologically combined control of GLI2 and ß-CATENIN effectively diminishes GSC properties. A ID1-high/CULLIN3-low expression signature correlates with a poor patient prognosis, supporting the clinical relevance of this signaling axis. Taken together, a loss of CULLIN3 represents a common signaling node for controlling the activity of intracellular WNT and SHH signaling pathways mediated by ID1.

MiR-200b is involved in Tgf-ß signaling to regulate mammalian palate development.

Various cellular and molecular events are involved in palatogenesis, including apoptosis, epithelial-mesenchymal transition (EMT), cell proliferation, and cell migration. Smad2 and Snail, which are well-known key mediators of the transforming growth factor beta (Tgf-ß) pathway, play a crucial role in the regulation of palate development. Regulatory effects of microRNA 200b (miR-200b) on Smad2 and Snail in palatogenesis have not yet been elucidated. The aim of this study is to determine the relationship between palate development regulators miR-200b and Tgf-ß-mediated genes. Expression of miR-200b, E-cadherin, Smad2, and Snail was detected in the mesenchyme of the mouse palate, while miR-200b was expressed in the medial edge epithelium (MEE) and palatal mesenchyme. After the contact of palatal shelves, miR-200b was no longer expressed in the mesenchyme around the fusion region. The binding activity of miR-200b to both Smad2 and Snail was examined using a luciferase assay. MiR-200b directly targeted Smad2 and Snail at both cellular and molecular levels. The function of miR-200b was determined by overexpression via a lentiviral vector in the palatal shelves. Ectopic expression of miR-200b resulted in suppression of these Tgf-ß-mediated regulators and changes of apoptosis and cell proliferation in the palatal fusion region. These results suggest that miR-200b plays a crucial role in regulating the Smad2, Snail, and in apoptosis during palatogenesis by acting as a direct non-coding, influencing factor. Furthermore, the molecular interactions between miR-200b and Tgf-ß signaling are important for proper palatogenesis and especially for palate fusion. Elucidating the mechanism of palatogenesis may aid the design of effective gene-based therapies for the treatment of congenital cleft palate.

Interactions between Shh, Sostdc1 and Wnt signaling and a new feedback loop for spatial patterning of the teeth.

Each vertebrate species displays specific tooth patterns in each quadrant of the jaw: the mouse has one incisor and three molars, which develop at precise locations and at different times. The reason why multiple teeth form in the jaw of vertebrates and the way in which they develop separately from each other have been extensively studied, but the genetic mechanism governing the spatial patterning of teeth still remains to be elucidated. Sonic hedgehog (Shh) is one of the key signaling molecules involved in the spatial patterning of teeth and other ectodermal organs such as hair, vibrissae and feathers. Sostdc1, a secreted inhibitor of the Wnt and Bmp pathways, also regulates the spatial patterning of teeth and hair. Here, by utilizing maternal transfer of 5E1 (an anti-Shh antibody) to mouse embryos through the placenta, we show that Sostdc1 is downstream of Shh signaling and suggest a Wnt-Shh-Sostdc1 negative feedback loop as a pivotal mechanism controlling the spatial patterning of teeth. Furthermore, we propose a new reaction-diffusion model in which Wnt, Shh and Sostdc1 act as the activator, mediator and inhibitor, respectively, and confirm that such interactions can generate the tooth pattern of a wild-type mouse and can explain the various tooth patterns produced experimentally.

Immunohistochemical localization of cytokeratins in the junctional region of ectoderm and endoderm.

Although tridermic species have two junctional regions of ectoderm and endoderm between their epidermis and digestive tract, we actually know little about these particular boundaries. Cytokeratins are the major intermediate filaments of epithelial cells and show a high degree of tissue specificity. Therefore, to characterize the epithelial cells in the junctional region of ectoderm and endoderm, we immunohistochemically examined the localization of cytokeratins 5, 7/17, 14, 18, Sox17, and alpha-fetoprotein (AFP) in the oropharyngeal and anorectal regions during the mouse gastrulation process. At embryonic day (E) 9.5, cytokeratins 5, 7/17, 14, and 18 were detected in all epithelial cells of the oropharyngeal region. At E12.5, cytokeratin 5-positive cells were not observed in the middle area of the oral cavity; however, the immunoreactivity was strong in the anterior and posterior areas. The immunoreaction of cytokeratins 18 was seen only in the middle and posterior areas of the oral mucosa. Cytokeratins 7/17 and 14 were localized in all areas of the oropharyngeal region. Sox17 and AFP, which are endodermal markers, were detected in the middle and posterior areas of the oral mucosa, but not in the anterior area. Moreover, this same localization pattern of cytokeratins also existed in the anorectal region of the E12.5 embryo, suggesting that the localization of cytokeratins and endodermal markers might give an implication for the boundary between ectoderm and endoderm. These results also suggest that these cytokeratins are useful molecules for monitoring the epithelial cell differentiation in the junctional region of the germ layers.

Function analysis of mesenchymal Bcor in tooth development by using RNA interference.

Teeth, an excellent model for studying organogenesis, develop from a series of epithelial-mesenchymal interactions that are mediated by a complex molecular network. Bcor (BCL-6 interacting corepressor) has recently been discovered, but little is known about its function in tooth development. Mutations in BCOR affect humans with oculofaciocardiodental syndrome, which is an X-linked dominant disorder with presumed male lethality and which comprises microphthalmia, congenital cataracts, radiculomegaly, and cardiac and digital abnormalities. In this study, the Bcor expression pattern has been intensively investigated during mouse molar development. Bcor is expressed in both dental epithelium and the mesenchyme at E11.5. To understand the function of Bcor, knockdown of Bcor has been examined by using lentivirus-mediated RNA interference. Silencing of Bcor expression in dental mesenchymal cells at E14.5 causes dentinogenesis defects and retardation of tooth root development. Thus, our results suggest that Bcor expressed in the mesenchyme plays crucial roles during early tooth development. The function of Bcor expressed in the epithelium remains to be elucidated.

MAPK mediates Hsp25 signaling in incisor development.

Rodent incisors are continuously growing teeth that include all stages of amelogenesis. Understanding amelogenesis requires investigations of the genes and their gene products control the ameloblast phenotype. One of the mechanisms related to tooth differentiation is mitogen-activated protein kinase (MAPK) signaling. The extracellular-signal regulated kinase (ERK)/mitogen-activated protein kinase kinase (MEK) cascade is associated with mechanisms that control the cell cycle and cell survival. However, the roles of cascades in incisor development remain to be determined. In this study, we investigated incisor development and growth in the mouse based on MAPK signaling. Moreover, heat-shock protein (Hsp)-25 is well known to be a useful marker of odontoblast differentiation. We used anisomycin (a protein-synthesis inhibitor that activates MAPKs) and U0126 (a MAPK inhibitor that blocks ERK1/2 phosphorylation) to examine the role of MAPKs in Hsp25 signaling in the development of the mouse incisor. We performed immunohistochemistry and in vitro culture using incisor tooth germ, and found that phospho-ERK (pERK), pMEK, and Hsp25 localized in developing incisor ameloblasts and anisomycin failed to produce incisor development. In addition, Western blotting results showed that anisomycin stimulated the phosphorylation of ERK, MEK, and Hsp25, and that some of these proteins were blocked by the U0126. These findings suggest that MAPK signals play important roles in incisor formation, differentiation, and development by mediating Hsp25 signaling.

WDNM1 is associated with differentiation and apoptosis of mammary epithelial cells.

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFalpha treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFalpha induced WDNM1 expression showed the NFkappaB-dependent mechanism since it's expression was abrogated in IkappaBalphaM (super-repressor of NFkappaB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFalpha-NFkappaB signal pathway, is associated with HC11 cell apoptosis.

Wnt11/Fgfr1b cross-talk modulates the fate of cells in palate development.

Various cellular and molecular events underlie the elevation and fusion of the developing palate that occurs during embryonic development. This includes convergent extension, where the medial edge epithelium is intercalated into the midline epithelial seam. We examined the expression patterns of Wnt11 and Fgfr1b - which are believed to be key factors in convergent extension - in mouse palate development. Wnt-11 overexpression and beads soaked in SU5402 (an Fgfr1 inhibitor) were employed in in vitro organ cultures. The results suggested that interactions between Wnt11 and Fgfr1b are important in modulating cellular events such as cell proliferation for growth and apoptosis for fusion. Moreover, the Wnt11 siRNA results showed that Wnt11-induced apoptosis was necessary for palatal fusion. In summary, Fgfr1b induces cell proliferation in the developing palate mesenchyme so that the palate grows and contacts each palatal shelf, with negative feedback of Fgfs triggered by excessive cell proliferation then inhibiting the expression of Fgfr1b and activating the expression of Wnt11 to fuse each palate by activating apoptosis.

Induction of serum amyloid A genes is associated with growth and apoptosis of HC11 mammary epithelial cells.

In this study, we examined the expression and functions of serum amyloid A (SAA) isoforms during apoptosis of HC11 mammary gland epithelial cells. Expression of SAA mRNAs and apoptosis were increased in HC11 cells by serum withdrawal and gradually decreased upon the addition of serum, or epidermal growth factor (EGF). TNFalpha treatment of HC11 cells also induced expression of SAA genes, and the effect on SAA1 and SAA2 expression was suppressed by treatment with MG132, and in cells transfected with a dominant negative mutant form of IkappaBalpha. Similar results were observed in response to interleukin-1 (IL-1), IL-6 and interferon gamma (IFNgamma). Furthermore, overexpression of the SAA1 and SAA2 isoforms suppressed growth and accelerated apoptosis of HC11 cells by increasing caspase 3/7 and caspase 8 activities, but the apoptotic effect of tumor necrosis factor alpha (TNFalpha) on HC11 cells was not enhanced. We found that expression of SAA1 and SAA2, but not SAA3, was regulated by an NFkappaB-dependent pathway, and that overexpression of SAA isoforms accelerated the apoptosis of HC11 cells.

Myogenic differentiation of p53- and Rb-deficient immortalized and transformed bovine fibroblasts in response to MyoD.

We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and p16(INK4a) functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and p16(INK4a) pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.

Establishment of immortal swine kidney epithelial cells.

Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.

Interferon regulatory factor 3 activates p53-dependent cell growth inhibition.

Interferon regulatory factor 3 (IRF3) is a transcriptional factor that plays a crucial role in activation of innate immunity and inflammation in response to viral infection. We investigated the biological function of IRF3 overexpressed in somatic cells such as fibroblasts and astrocytes. Similar to overexpression of oncogenic H-ras in the normal human fibroblast, overexpression of IRF3 in human fibroblast BJ cells was shown to decrease cell growth and increase senescence-associated beta-galactosidase activity by activating a p53 tumor suppressor. BCNU, a DNA damage agent, further accelerated p53 function and cell death in the IRF3-overexpressed BJ cells compared to control BJ cells, without increased expression of IRF3 target genes. IRF3 failed to activate p53 function and cell growth inhibition in BJ cells downregulating p53 by RNAi-mediated p53 knockdown. Furthermore, enforced expression of IRF3 did not show any effect of cell growth inhibition in astrocytes or embryonic fibroblasts derived from the p53(-/-) mouse. When compared to control BJ cells, BJ cells which downregulated IRF3 by RNAi-mediated IRF3 knockdown showed extended in vitro life span. Taken together, the present study indicates that IRF3 should be a novel inducer of cell growth inhibition and cellular senescence through activation of p53 tumor suppressor.

Establishment and characterization of three immortal bovine muscular epithelial cell lines.

We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.

Survivin inhibits anti-growth effect of p53 activated by aurora B.

Genomic instability and apoptosis evasion are hallmarks of cancer, but the molecular mechanisms governing these processes remain elusive. Here, we found that survivin, a member of the apoptosis-inhibiting gene family, and aurora B kinase, a chromosomal passenger protein, were co-overexpressed in the various glioblastoma cell lines and tumors. Notably, exogenous introduction of the aurora B in human BJ cells was shown to decrease cell growth and increase the senescence-associated beta-galactosidase activity by activation of p53 tumor suppressor. However, aurora B overexpression failed to inhibit cell proliferation in BJ and U87MG cells transduced with dominant-negative p53 as well as in p53(-/-) mouse astrocytes. Aurora B was shown to increase centrosome amplification in the p53(-/-) astrocytes. Survivin was shown to induce anchorage-independent growth and inhibit anti-proliferation and drug-sensitive apoptosis caused by aurora B. Overexpression of both survivin and aurora B further accelerated the proliferation of BJ cells. Taken together, the present study indicates that survivin should accelerate tumorigenesis by inhibiting the anti-proliferative effect of p53 tumor suppressor that is activated by aurora B in normal and glioblastoma cells containing intact p53.

Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells.

Although primary bovine embryonic fibroblast (BEF) cells have previously been used as nucleus-donors for nuclear transfer (NT), it has now been proposed to use BEF cells to generate cloned cows that were genetically modified by transgenic or a knock-out system. A major limitation to gene targeting somatic cells, however, is the overall life-span of the cell. In this study, we first examined in vitro life-span of primary BEF cells. Primary BEF cells were found to be replicative senescent at passage 10th-12th, similar to primary murine embryonic fibroblast cells. To overcome this short in vitro life-span, we have optimized culture conditions to extend the life-span and determined growth characteristics of BEF cell lines. Two life-span extended BEF cell lines (designated CGFR -BO-1 and CGFR-BO-2) were shown to grow much faster than their parental primary counterparts. Both cell lines did not display any potential for abnormal growth such as foci formations in either soft-agar or confluent culture condition. In cloning experiments using these cell lines as a nuclear donor, the reconstructed karyoblasts underwent apoptosis, reprogramming and development in the blastocyst stage, at a similar frequency to those observed with parental as well as adult primary fibroblasts. Furthermore, these cell lines targeted with green fluorescence protein (GFP) were successfully transduced, selected and reprogrammed by NT to develop into a blastocyst stage with GFP expression. Our results suggested methods to extend life-span of donor cells with tremendous implications for the genetic engineering of bovine fibroblast cells.