The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.
An asymptomatic 56-year-old woman, upon medical examination, was diagnosed with advanced adenocarcinoma of the upper third of the stomach. Computed tomography showed that the primary gastric tumor was directly invading the splenic hilum, and there were multiple metastases in the spleen; incurable gastric cancer was confirmed. S-1 plus cisplatin therapy was introduced as the induction regimen, and the main tumor and splenic metastases reduced significantly. Total gastrectomy with splenectomy and D2 lymphadenectomy was performed after 9 courses of chemotherapy. The surgery was completed with no residual tumor, and intraperitoneal washing cytology was negative. Histologically, the primary tumor was classified as Grade 2, reflecting the effect of chemotherapy, and viable metastatic tumors were confirmed in the spleen. S-1-based treatment was continued as adjuvant chemotherapy, and the patient was alive with no evidence of tumor recurrence 39 months after the initiation of chemotherapy. Although metastasis to the spleen from gastric adenocarcinoma has been reported as a rare metastatic pattern with poor prognosis, our patient had a long survival time after gastrectomy following induction chemotherapy.
Nanog, a core pluripotency factor, is required for stabilizing pluripotency of inner cell mass (ICM) and embryonic stem cells (ESCs), and survival of primordial germ cells in mice. Here, we have addressed function and regulation of Nanog in epiblasts of postimplantation mouse embryos by conditional knockdown (KD), chromatin immunoprecipitation (ChIP) using in vivo epiblasts, and protein interaction with the Nanog promoter in vitro. Differentiation of Nanog-KD epiblasts demonstrated requirement for Nanog in stabilization of pluripotency. Nanog expression in epiblast is directly regulated by Nodal/Smad2 pathway in a visceral endoderm-dependent manner. Notably, Nanog promoters switch from Oct4/Esrrb in ICM/ESCs to Oct4/Smad2 in epiblasts. Smad2 directly associates with Oct4 to form Nanog promoting protein complex. Collectively, these data demonstrate that Nanog plays a key role in stabilizing Epiblast pluripotency mediated by Nodal/Smad2 signaling, which is involved in Nanog promoter switching in early developing embryos.
In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca(2+)] change in the ?-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of ?-cells.
Aquatic organisms such as cichlids, coelacanths, seals, and cetaceans are active in UV-blue color environments, but many of them mysteriously lost their abilities to detect these colors. The loss of these functions is a consequence of the pseudogenization of their short wavelength-sensitive (SWS1) opsin genes without gene duplication. We show that the SWS1 gene (BdenS1?) of the deep-sea fish, pearleye (Benthalbella dentata), became a pseudogene in a similar fashion about 130million years ago (Mya) yet it is still transcribed. The rates of nucleotide substitution (~1.4×10(-9)/site/year) of the pseudogenes of these aquatic species as well as some prosimian and bat species are much smaller than the previous estimates for the globin and immunoglobulin pseudogenes.
Two cases of mucinous cystadenoma of the appendix successfully treated by laparoscopy are reported. An 81-year-old woman with lower right back pain was diagnosed with mucinous cystadenoma of the appendix or appendiceal carcinoma and underwent elective laparoscopic surgery. The other case involved a 70-year-old man with hematochezia who was diagnosed with mucinous cystadenoma. He also underwent elective laparoscopic surgery. In these two cases, gauze was folded around the tumors rather than grasping them directly. The postoperative courses were uneventful, and these patients had no recurrent disease at 2-year follow-up. In such cases, surgical excision of the tumor without rupture is of paramount importance because rupture of the lesion can cause pseudomyxoma peritonei. Though appendiceal mucinous cystadenoma has been considered a contraindication of laparoscopic resection, it was possible to achieve this by using a laparoscopic procedure with a gauze technique.
Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.
Conventional human induced pluripotent stem cells (hiPSCs), reprogrammed from somatic cells by induced expression of Oct4, Sox2, Klf4, and c-Myc, are phenotypically different from mouse embryonic stem cells (ESCs). In mice, culture in N2B27 serum-free 2i media (mitogen-activated protein kinase/extracellular signal-regulated kinase and glycogen synthase kinase 3 inhibitors; PD0325901 and CHIR99021) plus leukemia inhibitory factor (LIF) (2i+LIF medium) enriches for germline competent ESCs. Here, we demonstrate that flat-shaped hiPSC colonies can be reprogrammed into bowl-shaped multi-potent stem cells (2i-hiPSCs) by using 2i+LIF medium. Mechanical dissociation of 2i-hiPSC colonies enables stable maintenance for >20 passages. Importantly, gene expression profiling demonstrated that 2i-hiPSCs more closely resemble primitive neural stem cells (PNSCs). Notably, this 2i-induced phenotype was generated from conventional hiPSCs, but not human ESCs (hESCs), thus correlating with the observation of neuroectodermal SOX1-positive colonies in conventional hiPSCs, but not hESCs in 2i+LIF medium. Thus, 2i-hiPSCs, which are nonteratoma forming PNSCs, may represent a safe source of cells for neural research and regenerative medicine.
The synergetic effects of multiple rhodopsin mutations on color tuning need to be completely elucidated. Systematic genetic studies and spectroscopy have demonstrated an interesting example of synergetic color tuning between two amino acid residues in conger rhodopsins ancestral pigment (p501): -a double mutation at one nearby and one distant residue led to a significant ?(max) blue shift of 13 nm, whereas neither of the single mutations at these two sites led to meaningful shifts.To analyze the molecular mechanisms of this synergetic color tuning, we performed homology modeling, molecular simulations, and electronic state calculations. For the double mutant, N195A/A292S, in silico mutation analysis demonstrated conspicuous structural changes in the retinal chromophore, whereas that of the single mutant, A292S, was almost unchanged. Using statistical ensembles of QM/MM optimized structures, the excitation energy of retinal chromophore was evaluated for the three visual pigments. As a result, the ?(max) shift of double mutant (DM) from p501 was -8 nm, while that of single mutant (SM) from p501 was +1 nm. Molecular dynamics simulation for DM demonstrated frequent isomerization between 6-s-cis and 6-s-trans conformers. Unexpectedly, however, the two conformers exhibited almost identical excitation energy, whereas principal component analysis (PCA) identified the retinal-counterion cooperative change of BLA (bond length alternation) and retinal-counterion interaction lead to the shift.
Induced pluripotent stem cells (iPSCs) are generated by directly reprogramming somatic cells by forcing them to express the exogenous transcription factors, Oct4, Sox2, Klf4 and c-Myc (OSKM). These cells could potentially be used in clinical applications and basic research. Here, we explored the molecular role of Sox2 by generating iPSCs that expressed Sox2 at various levels. Low Sox2 (LS) expression increased the efficiency of generating partially reprogrammed iPSCs in combination with OKM. Notably, we detected a significant increase in the number of fully reprogrammed iPSCs with three factors of OK and LS. LS expression was linked with the reduced expression of ectoderm and mesoderm marker genes. This indicates that cell differentiation into the ectoderm and mesoderm lineages was impeded during reprogramming. The quality of the iPSCs that was generated by using OK and LS was comparable to that of iPSCs that were produced via conventional OSK as seen by pluripotent marker gene expression and chimera formation. We conclude that Sox2 plays a crucial role in a dose-dependent manner in direct reprogramming of somatic cells to iPSCs.
The lack of knowledge of current public attitudes towards basic research into induced pluripotent stem cells (iPSCs) is a serious problem when considering appropriate ways of governance regarding research and its clinical applications. We therefore conducted an internet-based survey to determine public opinion regarding the research and development of iPSCs and regenerative medicine (RM). A total of 14,908 valid responses were collected, which revealed that the Japanese public were familiar with the terms iPSCs and RM, and many of them had received information about iPSCs and RM through the television and newspapers. They also generally accepted the need for extra funding for research into iPSCs, but also decided to adopt a "wait and see" approach and thought that research and development of iPSCs and RM should be conducted under proper governance in accordance with an international regulatory framework. It will be necessary to discuss an internationally consistent regulatory system and effective mechanisms for information flow.
Practical clinical applications for current induced pluripotent stem cell (iPSC) technologies are hindered by very low generation efficiencies. Here, we demonstrate that newborn human (h) and mouse (m) extra-embryonic amnion (AM) and yolk-sac (YS) cells, in which endogenous KLF4/Klf4, c-MYC/c-Myc and RONIN/Ronin are expressed, can be reprogrammed to hiPSCs and miPSCs with efficiencies for AM cells of 0.02% and 0.1%, respectively. Both hiPSC and miPSCs are indistinguishable from embryonic stem cells in colony morphology, expression of pluripotency markers, global gene expression profile, DNA methylation status of OCT4 and NANOG, teratoma formation and, in the case of miPSCs, generation of germline transmissible chimeric mice. As copious amounts of human AM cells can be collected without invasion, and stored long term by conventional means without requirement for in vitro culture, they represent an ideal source for cell banking and subsequent on demand generation of hiPSCs for personal regenerative and pharmaceutical applications.
The pluripotency factor Nanog is expressed in peri-implantation embryos and primordial germ cells (PGCs). Nanog-deficient mouse embryos die soon after implantation. To explore the function of Nanog in germ cells, Nanog RNA was conditionally knocked down in vivo by shRNA. Nanog shRNA transgenic (NRi-Tg) mice were generated through the formation of germline chimeras with NRi-Tg embryonic stem cells. In E12.5 Cre-induced ER-Cre/NRi-Tg and TNAP-Cre/NRi-Tg double-transgenic embryos, the number of alkaline phosphatase-positive and SSEA1-positive PGCs decreased significantly. In the E9.5 and E10.5 migrating Nanog-knockdown PGCs, TUNEL-positive apoptotic cell death became prominent in vivo and in vitro, despite Oct4 expression. Single-cell microarray analysis of E10.5 Nanog-knockdown PGCs revealed significant up- and downregulation of a substantial number of genes, including Tial1, Id1 and Suz12. These data suggest that Nanog plays a key role in the proliferation and survival of migrating PGCs as a safeguard of the PGC-specific molecular network.
The vertebrate ancestor possessed ultraviolet (UV) vision and many species have retained it during evolution. Many other species switched to violet vision and, then again, some avian species switched back to UV vision. These UV and violet vision are mediated by short wavelength-sensitive (SWS1) pigments that absorb light maximally (lambda(max)) at approximately 360 and 390-440 nm, respectively. It is not well understood why and how these functional changes have occurred. Here, we cloned the pigment of scabbardfish (Lepidopus fitchi) with a lambda(max) of 423 nm, an example of violet-sensitive SWS1 pigment in fish. Mutagenesis experiments and quantum mechanical/molecular mechanical (QM/MM) computations show that the violet-sensitivity was achieved by the deletion of Phe-86 that converted the unprotonated Schiff base-linked 11-cis-retinal to a protonated form. The finding of a violet-sensitive SWS1 pigment in scabbardfish suggests that many other fish also have orthologous violet pigments. The isolation and comparison of such violet and UV pigments in fish living in different ecological habitats will open an unprecedented opportunity to elucidate not only the molecular basis of phenotypic adaptations, but also the genetics of UV and violet vision.
Chromosomal deletions are widely involved in serious genetic diseases and in the pathogenesis of cancers. These deletions often generate loss of heterozygosity (LOH) of one of the alleles of a tumor suppressor gene. Because of the technical difficulty inherent in genetic manipulation studies of a chromosome-wide deficiency, it has not been experimentally determined whether chromosome deletions could be a trigger for cancer development. Using the Cre/inverted loxP system, we have developed a chromosome elimination cassette (CEC) that Cre-dependently induces whole or partial deletions of the CEC-tagged chromosomes. Most deletions are usually fatal, but diploid cells carrying small deletions have been obtained from mouse embryonic stem cells carrying a CEC transgene (CEC-ESC). Here, we further isolated various CEC-ESC clones and analyzed CEC integration sites using the fluorescence in-situ hybridization method. In 17 CEC-ESC clones possessing normal chromosome sets, 13 types of chromosomes out of 20 pairs of mouse chromosomes were tagged by CEC. Each CEC-tagged chromosome could become a future target for the creation of a Cre-inducible LOH by a combination of in vitro and in vivo genetic mutation.
Calcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria) and comb jellies (Phylum Ctenophora). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis.
Human induced pluripotent stem cells (iPSCs) are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs) as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC) lines through re-plating followed by embryoid body formation and serial transplantation. iCSCs shared the expression of pluripotent marker genes with iPSCs, except for REX1 and LIN28, while exhibited the expression of somatic marker genes EMP1 and PPAR?. iESCs and iCSCs could generate teratomas with high efficiency by implantation into immunodeficient mice. The second iCSCs isolated from dissociated cells of teratoma from the first iCSCs were stably maintained, showing a gene expression profile similar to the first iCSCs. In the first and second iCSCs, transgene-derived Oct4, Sox2, Klf4, and c-Myc were expressed. Comparative global gene expression analyses demonstrated that the first iCSCs were similar to iESCs, and clearly different from human iPSCs and somatic cells. In iCSCs, gene expression kinetics of the core pluripotency factor and the Myc-related factor were pluripotent type, whereas the polycomb complex factor was somatic type. These findings indicate that pluripotent tumorigenicity can be conferred on somatic cells through up-regulation of the core pluripotency and Myc-related factors, prior to establishment of the iPSC molecular network by full reprogramming through down-regulation of the polycomb complex factor.
In mouse embryos, some primordial germ cells (PGCs) are eliminated by apoptosis, but the molecular pathways that lead to PGC survival versus apoptosis have not been fully characterized. Here, we found that REST (repressor element 1-silencing transcription factor), a transcription factor that binds a conserved regulatory element, NRSE/RE1, played a role in PGC survival. REST expression was higher in PGCs than in surrounding somatic cells. Moreover, in mouse embryos with a PGC-specific conditional REST mutation, the PGC population experienced more apoptosis and was significantly smaller than that in control embryos; these findings indicated that REST functioned in a cell-autonomous fashion that was critical for PGC survival. Several anti-apoptotic genes were among the previously identified REST-target gene candidates; moreover, some of these genes were downregulated in the REST-deficient PGCs. Mek5, which encodes a component in the a MAP kinase cascade, was one of these downregulated REST-target gene candidates, and a Mek5 mutation, like the REST mutation, caused an increase in PGC apoptosis; these finding suggested that REST promoted PGC survival via regulation of the Mek5 expression. Importantly, there were a normal number of PGCs in the REST mutants at birth, and both the male and female REST-mutant adults were fertile; these final observations revealed that the PGC population was very robust and could recover from a genetically induced reduction in cell number.
The discovery of induced pluripotent stem (iPS) cells has broadened the promises of regenerative medicine through the generation of syngeneic replacement cells or tissues via the differentiation of patient-specific iPS cells. To apply iPS cell-mediated therapy to patients with genetic disorders, however, genome-editing technologies with high efficiency and specificity are needed. Recently, several targeted genome-editing strategies mediated by zinc finger nuclease and transcription activator-like effector nuclease have been applied to human and mouse iPS cells. Furthermore, spontaneous homologous recombination can restore genotype to wild type in mouse iPS cells heterozygous for genetic mutations. Through genome editing, the clinical application of patient-specific genetic mutation-free iPS cells to genetic disorders can finally be realized.
Somatic cell hybridization is widely used to study the control of gene regulation and the stability of differentiated states. In contrast, the application of this method to germ cells has been limited in part because of an inability to culture germ cells. In this study, we produced germ cell hybrids using germ-line stem (GS) cells and multipotent germ-line stem (mGS) cells. While GS cells are enriched for spermatogonial stem cell (SSC) activity, mGS cells are similar to embryonic stem (ES) cells and originally derived from GS cells. Hybrids were successfully obtained between GS cells and ES cells, between GS cells and mGS cells, and between mGS cells and thymocytes. All exhibited ES cell markers and a behavior similar to ES cells, formed teratomas, and differentiated into somatic cell tissues. However, none of the hybrid cells were able to reconstitute spermatogenesis after microinjection into seminiferous tubules. Analyses of the DNA methylation patterns of imprinted genes also showed that mGS cells do not possess a DNA demethylation ability, which was found in embryonic germ cells derived from primordial germ cells. However, mGS cells reactivated the X chromosome and induced Pou5f1 expression in female thymocytes in a manner similar to ES cells. These data show that mGS cells possess ES-like reprogramming potential, which predominates over-SSC activity.
Induced pluripotent stem cells (iPSCs) generated by epigenetic reprogramming of personal somatic cells have limited therapeutic capacity for patients suffering from genetic disorders. Here we demonstrate restoration of a genomic mutation heterozygous for Pkd1 (polycystic kidney disease 1) deletion (Pkd1(+/-) to Pkd1(+/R+)) by spontaneous mitotic recombination. Notably, recombination between homologous chromosomes occurred at a frequency of 1~2 per 10,000 iPSCs. Southern blot hybridization and genomic PCR analyses demonstrated that the genotype of the mutation-restored iPSCs was indistinguishable from that of the wild-type cells. Importantly, the frequency of cyst generation in kidneys of adult chimeric mice containing Pkd1(+/R+) iPSCs was significantly lower than that of adult chimeric mice with parental Pkd1(+/-) iPSCs, and indistinguishable from that of wild-type mice. This repair step could be directly incorporated into iPSC development programmes prior to cell transplantation, offering an invaluable step forward for patients carrying a wide range of genetic disorders.
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