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Sample records for cells hescs grown

  1. Effect of ROCK inhibitor Y-27632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion.

    PubMed

    Gauthaman, Kalamegam; Fong, Chui-Yee; Bongso, Ariff

    2010-03-01

    The Rho associated coiled coil protein kinase (ROCK) dependent signaling pathway plays an important role in numerous physiological functions such as cell proliferation, adhesion, migration and inflammation. Human embryonic stem cells (hESCs) undergo differentiation and poor survival after single cell dissociation in culture thus limiting their expansion for cell based therapies. We evaluated the role of the selective ROCK inhibitor Y-27632 on hESC colonies and disassociated single hESCs from two different hESC lines. Karyotypically normal hESCs (HES3) and variant hESCs (BG01V) were treated with Y-27632 at 5, 10 and 20 muM concentrations for 72 h and its effects on hESC self renewal, colony morphology, cell cycle and pluripotency were evaluated. Increased cell proliferation of both HES3 and BG01V were observed for all three concentrations compared to untreated controls following passaging of cell clusters or dissociated single cells and some of these increases were statistically significant. Cell cycle assay demonstrated normal cell cycle progression with no peaks evident of apoptosis. No morphological differentiation was evident following treatment with the highest concentration of Y-27632 (20 muM) and the stemness related genes continued to be highly expressed in both HES3 and BG01V cells compared to untreated controls. The results confirmed that Y-27632 is a useful agent that aids in the expansion of undifferentiated hESC numbers for downstream applications in regenerative medicine.

  2. Roles of hesC and gcm in echinoid larval mesenchyme cell development.

    PubMed

    Yamazaki, Atsuko; Minokawa, Takuya

    2016-04-01

    To understand the roles of hesC and gcm during larval mesenchyme specification and differentiation in echinoids, we performed perturbation experiments for these genes in two distantly related euechinoids, Hemicentrotus pulcherrimus and Scaphechinus mirabilis. The number of larval mesenchyme cells increased when the translation of hesC was inhibited, thereby suggesting that hesC has a general role in larval mesenchyme development. We confirmed previous results by demonstrating that gcm is involved in pigment cell differentiation. Simultaneous inhibition of the translation of hesC and gcm induced a significant increase in the number of skeletogenic cells, which suggests that gcm functions in skeletogenic fate repression. Based on these observations, we suggest that: (i) hesC participates in some general aspects of mesenchymal cell development; and (ii) gcm is involved in the mechanism responsible for the binary specification of skeletogenic and pigment cell fates. PMID:27046223

  3. Donating embryos for human embryonic stem cell (hESC) research: a committee opinion.

    PubMed

    2013-10-01

    hESC research is an ethically acceptable use of human embryos that are in excess of those needed to meet the fertility goals of patients. The ethical basis for this view and issues to be considered during the informed consent process for the donation of embryos are developed in this document. This report replaces the Committee's 2009 report, "Donating spare embryos for stem cell research" (Fertil Steril 2009;91:667-70).

  4. Label-free separation of human embryonic stem cells (hESCs) and their cardiac derivatives using Raman spectroscopy

    SciTech Connect

    Chan, J W; Lieu, D K; Huser, T R; Li, R A

    2008-09-08

    Self-renewable, pluripotent human embryonic stem cells (hESCs) can be differentiated into cardiomyocytes (CMs), providing an unlimited source of cells for transplantation therapies. However, unlike certain cell lineages such as hematopoietic cells, CMs lack specific surface markers for convenient identification, physical separation, and enrichment. Identification by immunostaining of cardiac-specific proteins such as troponin requires permeabilization, which renders the cells unviable and non-recoverable. Ectopic expression of a reporter protein under the transcriptional control of a heart-specific promoter for identifying hESC-derived CMs (hESC-CMs) is useful for research but complicates potential clinical applications. The practical detection and removal of undifferentiated hESCs in a graft, which may lead to tumors, is also critical. Here, we demonstrate a non-destructive, label-free optical method based on Raman scattering to interrogate the intrinsic biochemical signatures of individual hESCs and their cardiac derivatives, allowing cells to be identified and classified. By combining the Raman spectroscopic data with multivariate statistical analysis, our results indicate that hESCs, human fetal left ventricular CMs, and hESC-CMs can be identified by their intrinsic biochemical characteristics with an accuracy of 96%, 98% and 66%, respectively. The present study lays the groundwork for developing a systematic and automated method for the non-invasive and label-free sorting of (i) high-quality hESCs for expansion, and (ii) ex vivo CMs (derived from embryonic or adult stem cells) for cell-based heart therapies.

  5. Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines

    PubMed Central

    Venkatesh, Priyanka; Panyutin, Irina V.; Remeeva, Evgenia; Neumann, Ronald D.; Panyutin, Igor G.

    2016-01-01

    Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC. PMID:26729112

  6. Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines.

    PubMed

    Venkatesh, Priyanka; Panyutin, Irina V; Remeeva, Evgenia; Neumann, Ronald D; Panyutin, Igor G

    2016-01-02

    Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.

  7. Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies.

    PubMed

    Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

    2016-01-01

    Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

  8. Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

    PubMed Central

    Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

    2016-01-01

    Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

  9. Human embryonic stem cells (hESCs) in the treatment of emphysematous COPD: a case report

    PubMed Central

    Shroff, Geeta

    2015-01-01

    Key Clinical Message Emphysema results in narrowing of the small airways due to inhaling of cigarette smoke and other noxious particles. Oxygen therapy, corticosteroids, and bronchodilators increase the risk of pneumonia, arrhythmia, and fractures in long term. Therapy with human embryonic stem cells resulted in improved symptoms of a patient with emphysema. PMID:26273458

  10. MAFA and T3 Drive Maturation of Both Fetal Human Islets and Insulin-Producing Cells Differentiated From hESC

    PubMed Central

    Aguayo-Mazzucato, Cristina; DiIenno, Amanda; Hollister-Lock, Jennifer; Cahill, Christopher; Sharma, Arun; Weir, Gordon; Colton, Clark

    2015-01-01

    Context: Human embryonic stem cells (hESCs) differentiated toward β-cells and fetal human pancreatic islet cells resemble each other transcriptionally and are characterized by immaturity with a lack of glucose responsiveness, low levels of insulin content, and impaired proinsulin-to-insulin processing. However, their response to stimuli that promote functionality have not been compared. Objective: The objective of the study was to evaluate the effects of our previous strategies for functional maturation developed in rodents in these two human models of β-cell immaturity and compare their responses. Design, Settings, Participants, and Interventions: In proof-of-principle experiments using either adenoviral-mediated overexpression of V-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA) or the physiologically driven path via thyroid hormone (T3) and human fetal islet-like cluster (ICC) functional maturity was evaluated. Then the effects of T3 were evaluated upon the functional maturation of hESCs differentiated toward β-cells. Main Outcome Measures: Functional maturation was evaluated by the following parameters: glucose responsiveness, insulin content, expression of the mature β-cell transcription factor MAFA, and proinsulin-to-insulin processing. Results: ICCs responded positively to MAFA overexpression and T3 treatment as assessed by two different maturation parameters: increased insulin secretion at 16.8 mM glucose and increased proinsulin-to-insulin processing. In hESCs differentiated toward β-cells, T3 enhanced MAFA expression, increased insulin content (probably mediated by the increased MAFA), and increased insulin secretion at 16.8 mM glucose. Conclusion: T3 is a useful in vitro stimulus to promote human β-cell maturation as shown in both human fetal ICCs and differentiated hESCs. The degree of maturation induced varied in the two models, possibly due to the different developmental status at the beginning of the study. PMID:26207953

  11. Impact of transient down-regulation of DREAM in human embryonic stem cell pluripotency: The role of DREAM in the maintenance of hESCs.

    PubMed

    Fontán-Lozano, A; Capilla-Gonzalez, V; Aguilera, Y; Mellado, N; Carrión, A M; Soria, B; Hmadcha, A

    2016-05-01

    Little is known about the functions of downstream regulatory element antagonist modulator (DREAM) in embryonic stem cells (ESCs). However, DREAM interacts with cAMP response element-binding protein (CREB) in a Ca(2+)-dependent manner, preventing CREB binding protein (CBP) recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAM depletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs) function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs), human adipose-derived stromal cells (hASCs), human bone marrow-derived stromal cells (hBMSCs), and human newborn foreskin fibroblasts (hFFs), and that transitory inhibition of DREAM in hESCs reduces their pluripotency, increasing differentiation. We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAM acts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells. PMID:26999760

  12. Impact of transient down-regulation of DREAM in human embryonic stem cell pluripotency: The role of DREAM in the maintenance of hESCs.

    PubMed

    Fontán-Lozano, A; Capilla-Gonzalez, V; Aguilera, Y; Mellado, N; Carrión, A M; Soria, B; Hmadcha, A

    2016-05-01

    Little is known about the functions of downstream regulatory element antagonist modulator (DREAM) in embryonic stem cells (ESCs). However, DREAM interacts with cAMP response element-binding protein (CREB) in a Ca(2+)-dependent manner, preventing CREB binding protein (CBP) recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAM depletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs) function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs), human adipose-derived stromal cells (hASCs), human bone marrow-derived stromal cells (hBMSCs), and human newborn foreskin fibroblasts (hFFs), and that transitory inhibition of DREAM in hESCs reduces their pluripotency, increasing differentiation. We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAM acts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells.

  13. Fresh embryo donation for human embryonic stem cell (hESC) research: the experiences and values of IVF couples asked to be embryo donors

    PubMed Central

    Haimes, E.; Taylor, K.

    2009-01-01

    BACKGROUND This article reports on an investigation of the views of IVF couples asked to donate fresh embryos for research and contributes to the debates on: the acceptability of human embryonic stem cell (hESC) research, the moral status of the human embryo and embryo donation for research. METHODS A hypothesis-generating design was followed. All IVF couples in one UK clinic who were asked to donate embryos in 1 year were contacted 6 weeks after their pregnancy result. Forty four in-depth interviews were conducted. RESULTS Interviewees were preoccupied with IVF treatment and the request to donate was a secondary consideration. They used a complex and dynamic system of embryo classification. Initially, all embryos were important but then their focus shifted to those that had most potential to produce a baby. At that point, ‘other’ embryos were less important though they later realise that they did not know what happened to them. Guessing that these embryos went to research, interviewees preferred not to contemplate what that might entail. The embryos that caused interviewees most concern were good quality embryos that might have produced a baby but went to research instead. ‘The’ embryo, the morally laden, but abstract, entity, did not play a central role in their decision-making. CONCLUSIONS This study, despite missing those who refuse to donate embryos, suggests that debates on embryo donation for hESC research should include the views of embryo donors and should consider the social, as well as the moral, status of the human embryo. PMID:19502616

  14. Factors expressed by murine embryonic pancreatic mesenchyme enhance generation of insulin-producing cells from hESCs.

    PubMed

    Guo, Tingxia; Landsman, Limor; Li, Na; Hebrok, Matthias

    2013-05-01

    Islet transplantation has proven to be a successful strategy to restore normoglycemia in patients with type 1 diabetes (T1D). However, the dearth of cadaveric islets available for transplantation hampers the widespread application of this treatment option. Although human embryonic stem cells and induced pluripotent stem cells are capable of generating insulin-producing cells in vitro when provided with the appropriate inductive cues, the insulin-expressing cells that develop behave more like immature β-cells with minimal sensitivity to glucose stimulation. Here, we identify a set of signaling factors expressed in mouse embryonic mesenchyme during the time when foregut and pancreatic progenitors are specified and test their activities during in vitro differentiation of human embryonic stem cells. Several of the identified factors work in concert to expand the pancreatic progenitor pool. Interestingly, transforming growth factor (TGF)-β ligands, most potent in inducing pancreatic progenitors, display strong inhibitory effects on subsequent endocrine cell differentiation. Treatment with TGF-β ligands, followed by the addition of a TGF-β receptor antagonist, dramatically increased the number of insulin-producing cells in vitro, demonstrating the need for dynamic temporal regulation of TGF-β signaling during in vitro differentiation. These studies illustrate the need to precisely mimic the in vivo conditions to fully recapitulate pancreatic lineage specification in vitro. PMID:23305648

  15. hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature

    PubMed Central

    Acevedo, Lisette M.; Lindquist, Jeffrey N.; Walsh, Breda M.; Sia, Peik; Cimadamore, Flavio; Chen, Connie; Denzel, Martin; Pernia, Cameron D.; Ranscht, Barbara; Terskikh, Alexey; Snyder, Evan Y.; Cheresh, David A.

    2015-01-01

    Summary To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC), not the neural tube (NT). Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning. PMID:26004631

  16. Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm.

    PubMed

    Chayosumrit, Methichit; Tuch, Bernard; Sidhu, Kuldip

    2010-01-01

    Human embryonic stem cells (hESCs) are potential renewable sources of cells in replacement therapies for many diseases including type 1 diabetes. We have established a three dimensional (3D) model to culture and differentiate hESCs that are encapsulated in calcium alginate microcapsules. This system promotes cellular interactions that are essential for both maintaining pluripotency and differentiation. This 3D model also provides opportunity to separate out hESCs from fibroblasts used as feeder layer during culture. In this study, we compared the viability and proliferation of the encapsulated hESCs cultured in serum replacement (SR) medium, human fetal fibroblast-conditioned medium (hFF-CM), in the presence and absence of Y-27632, a ROCK inhibitor. Treatment of hESCs with Y-27632 promoted cell survival, cell cluster formation and proliferation rate in both SR medium and hFF-CM. These encapsulated hESC clusters were then directly differentiated to definitive endoderm cells that expressed mesendoderm (Brachyury 70-fold), definitive endoderm (SOX17>300-fold, FOXA2>800-fold, and CXCR4>100-fold) and primitive gut tube (HNF1beta>120-fold) as compared to the undifferentiated hESCs. These data show that microcapsules can be used for differentiation of hESCs into definitive endoderm in 3D and could have potential application for immune-isolation and prevention of teratomas formation of hESCs during transplantation. PMID:19833385

  17. Fibrochondrogenesis of hESCs: growth factor combinations and cocultures.

    PubMed

    Hoben, Gwendolyn M; Willard, Vincent P; Athanasiou, Kyriacos A

    2009-03-01

    The successful differentiation of human embryonic stem cells (hESCs) to fibrochondrocyte-like cells and characterization of these differentiated cells is a critical step toward tissue engineering of musculoskeletal fibrocartilages (e.g., knee meniscus, temporomandibular joint disc, and intervertebral disc). In this study, growth factors and primary cell cocultures were applied to hESC embryoid bodies (EBs) for 3 weeks and evaluated for their effect on the synthesis of critical fibrocartilage matrix components: glycosaminoglycans (GAG) and collagens (types I, II, and VI). Changes in surface markers (CD105, CD44, SSEA, PDGFR alpha) after the differentiation treatments were also analyzed. The study was conducted in three phases: (1) examination of growth factors (TGF-beta 3, BMP-2, BMP-4, BMP-6, PDGF-BB, sonic hedgehog protein); (2) comparison of two cocultures (primary chondrocytes or fibrochondrocytes); and (3) the combination of the most effective growth factor and coculture regimen. TGF-beta 3 with BMP-4 yielded EBs positive for collagens I, II, and VI, with up to 6.7- and 4.8-fold increases in GAG and collagen, respectively. Analysis of cell surface markers showed a significant increase in CD44 with the TGF-beta 3 + BMP-4 treatment compared to the controls. Coculture with fibrochondrocytes resulted in up to a 9.8-fold increase in collagen II production. The combination of the growth factors BMP-4 + TGF-beta 3 with the fibrochondrocyte coculture led to an increase in cell proliferation and GAG production compared to either treatment alone. This study determined two powerful treatments for inducing fibrocartilaginous differentiation of hESCs and provides a foundation for using flow cytometry to purify these differentiated cells. PMID:18454697

  18. Genome-Wide Transcriptome and Binding Sites Analyses Identify Early FOX Expressions for Enhancing Cardiomyogenesis Efficiency of hESC Cultures.

    PubMed

    Yeo, Hock Chuan; Ting, Sherwin; Brena, Romulo Martin; Koh, Geoffrey; Chen, Allen; Toh, Siew Qi; Lim, Yu Ming; Oh, Steve Kah Weng; Lee, Dong-Yup

    2016-01-01

    The differentiation efficiency of human embryonic stem cells (hESCs) into heart muscle cells (cardiomyocytes) is highly sensitive to culture conditions. To elucidate the regulatory mechanisms involved, we investigated hESCs grown on three distinct culture platforms: feeder-free Matrigel, mouse embryonic fibroblast feeders, and Matrigel replated on feeders. At the outset, we profiled and quantified their differentiation efficiency, transcriptome, transcription factor binding sites and DNA-methylation. Subsequent genome-wide analyses allowed us to reconstruct the relevant interactome, thereby forming the regulatory basis for implicating the contrasting differentiation efficiency of the culture conditions. We hypothesized that the parental expressions of FOXC1, FOXD1 and FOXQ1 transcription factors (TFs) are correlative with eventual cardiomyogenic outcome. Through WNT induction of the FOX TFs, we observed the co-activation of WNT3 and EOMES which are potent inducers of mesoderm differentiation. The result strengthened our hypothesis on the regulatory role of the FOX TFs in enhancing mesoderm differentiation capacity of hESCs. Importantly, the final proportions of cells expressing cardiac markers were directly correlated to the strength of FOX inductions within 72 hours after initiation of differentiation across different cell lines and protocols. Thus, we affirmed the relationship between early FOX TF expressions and cardiomyogenesis efficiency. PMID:27501774

  19. Genome-Wide Transcriptome and Binding Sites Analyses Identify Early FOX Expressions for Enhancing Cardiomyogenesis Efficiency of hESC Cultures

    PubMed Central

    Yeo, Hock Chuan; Ting, Sherwin; Brena, Romulo Martin; Koh, Geoffrey; Chen, Allen; Toh, Siew Qi; Lim, Yu Ming; Oh, Steve Kah Weng; Lee, Dong-Yup

    2016-01-01

    The differentiation efficiency of human embryonic stem cells (hESCs) into heart muscle cells (cardiomyocytes) is highly sensitive to culture conditions. To elucidate the regulatory mechanisms involved, we investigated hESCs grown on three distinct culture platforms: feeder-free Matrigel, mouse embryonic fibroblast feeders, and Matrigel replated on feeders. At the outset, we profiled and quantified their differentiation efficiency, transcriptome, transcription factor binding sites and DNA-methylation. Subsequent genome-wide analyses allowed us to reconstruct the relevant interactome, thereby forming the regulatory basis for implicating the contrasting differentiation efficiency of the culture conditions. We hypothesized that the parental expressions of FOXC1, FOXD1 and FOXQ1 transcription factors (TFs) are correlative with eventual cardiomyogenic outcome. Through WNT induction of the FOX TFs, we observed the co-activation of WNT3 and EOMES which are potent inducers of mesoderm differentiation. The result strengthened our hypothesis on the regulatory role of the FOX TFs in enhancing mesoderm differentiation capacity of hESCs. Importantly, the final proportions of cells expressing cardiac markers were directly correlated to the strength of FOX inductions within 72 hours after initiation of differentiation across different cell lines and protocols. Thus, we affirmed the relationship between early FOX TF expressions and cardiomyogenesis efficiency. PMID:27501774

  20. Modeling Type 2 Diabetes GWAS Candidate Gene Function in hESCs.

    PubMed

    Rutter, Guy A

    2016-09-01

    Type 2 diabetes is a complex polygenic disorder that affects about 1 in 12 adults. In this issue of Cell Stem Cell, Zeng et al. (2016) elegantly combine CRISPR-based gene editing in hESCs with directed β cell differentiation to investigate the functions of genes highlighted by genome-wide association studies (GWAS) for this disease. PMID:27588741

  1. Colon tumor cells grown in NASA Bioreactor

    NASA Technical Reports Server (NTRS)

    2001-01-01

    These photos compare the results of colon carcinoma cells grown in a NASA Bioreactor flown on the STS-70 Space Shuttle in 1995 flight and ground control experiments. The cells grown in microgravity (left) have aggregated to form masses that are larger and more similar to tissue found in the body than the cells cultured on the ground (right). The principal investigator is Milburn Jessup of the University of Texas M. D. Anderson Cancer Center. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Cell constructs grown in a rotating bioreactor on Earth (left) eventually become too large to stay suspended in the nutrient media. In the microgravity of orbit, the cells stay suspended. Rotation then is needed for gentle stirring to replenish the media around the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Credit: NASA and University of Texas M. D. Anderson Cancer Center.

  2. ELABELA Is an Endogenous Growth Factor that Sustains hESC Self-Renewal via the PI3K/AKT Pathway.

    PubMed

    Ho, Lena; Tan, Shawn Y X; Wee, Sheena; Wu, Yixuan; Tan, Sam J C; Ramakrishna, Navin B; Chng, Serene C; Nama, Srikanth; Szczerbinska, Iwona; Sczerbinska, Iwona; Chan, Yun-Shen; Avery, Stuart; Tsuneyoshi, Norihiro; Ng, Huck Hui; Gunaratne, Jayantha; Dunn, N Ray; Reversade, Bruno

    2015-10-01

    ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFβ pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency.

  3. Enhancement of the propagation of human embryonic stem cells by modifications in the gel architecture of PMEDSAH polymer coatings

    PubMed Central

    Qian, Xu; Villa-Diaz, Luis G.; Kumar, Ramya; Lahann, Joerg; Krebsbach, Paul H.

    2014-01-01

    Well-defined culture conditions are essential for realizing the full potential of human embryonic stem cells (hESCs) in regenerative medicine where large numbers of cells are required. Synthetic polymers, such as poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] (PMEDSAH), offer multiple advantages over mouse embryonic fibroblasts (MEFs) and Matrigel™ for hESC culture and expansion. However, there is limited understanding of the mechanisms by which hESCs are propagated on synthetic polymers coatings. Here, the effects of PMEDSAH gel architecture on hESC self-renewal were determined. By increasing the atom transfer radical polymerization (ATRP) reaction time, the thickness of PMEDSAH was increased and its internal hydrogel architecture was modified, while maintaining its overall chemical structure. A 105 nm thick ATRP PMEDSAH coating showed a significant increase in the expansion rate of hESCs. Theoretical calculations suggested that 20,000 hESCs cultured on this substrate could be expanded up to 4.7×109 undifferentiated cells in five weeks. In addition, hESCs grown on ATRP PMEDSAH coatings retained pluripotency and displayed a normal karyotype after long-term culture. These data demonstrate the importance of polymer physical properties in hESC expansion. This and similar modifications of PMEDSAH coatings may be used to obtain large populations of hESCs required for many applications in regenerative medicine. PMID:25189518

  4. Cell-mediated destruction of cells grown on artificial capillaries.

    PubMed

    Zwilling, B S; Clayman, D A

    1978-11-01

    This investigation was designed to determine the conditions required to assess cell-mediated destruction of target L-cells grown on artificial capillaries. In control cultures that contained L-cells alone, solid nodules with a diameter of 1 mm as well as dense cellular growth could be visually observed by the 12th day of culture. Alloimmune spleen cells from both immunized and normal C57BL/10 mice were shown to be capable of destroying tritiated thymidine-labeled L-cells growing on artificial capillaries. The destruction of target cells grown as monolayers in capillary culture correlated well with monolayer cultures incubated in 16-mm plastic tissue culture wells. When target cells were grown in capillary culture for 5 days before the addition of effector cells, significant destruction by normal effector cells was not observed until the 15th day of culture whereas that mediated by immune cells was observed by the 7th day. The possible effects of cell-culturing conditions on the kinetics of cell-mediated destruction in capillary chambers are discussed.

  5. Derivation, culture, and characterization of VUB hESC lines.

    PubMed

    Mateizel, Ileana; Spits, Claudia; De Rycke, Martine; Liebaers, Inge; Sermon, Karen

    2010-04-01

    In this report, we present the derivation and characterization of 15 hESC lines established at the Vrije Universiteit Brussel, Belgium in collaboration with the Universitair Ziekenhuis Brussel, Belgium, using surplus in vitro fertilization embryos and embryos carrying monogenic disorders donated for research. Four lines were derived from blastocyst-stage embryos presumed to be genetically normal, and 11 hESC lines were obtained from embryos shown to carry genetic mutations by preimplantation genetic diagnosis. All the lines express markers of pluripotency as determined by immunocytochemistry and RT-PCR, and formed teratomas when injected into SCID mice. All VUB hESC lines, except for VUB17, are reported in the European hESC registry and are available upon request after signing a Material Transfer Agreement from the VUB (contact person: Prof. Dr. Karen Sermon; Karen.Sermon@uzbrussel.be). PMID:20224973

  6. X-Inactivation Reveals Epigenetic Anomalies in Most hESC but Identifies Sublines That Initiate as Expected

    PubMed Central

    HALL, LISA L.; BYRON, MEG; BUTLER, JOHN; BECKER, KLAUS A.; NELSON, ANGEL; AMIT, MICHAL; ITSKOVITZ-ELDOR, JOSEPH; STEIN, JANET; STEIN, GARY; WARE, CAROL; LAWRENCE, JEANNE B.

    2011-01-01

    The clinical and research value of human embryonic stem cells (hESC) depends upon maintaining their epigenetically naïve, fully undifferentiated state. Inactivation of one X chromosome in each cell of mammalian female embryos is a paradigm for one of the earliest steps in cell specialization through formation of facultative heterochromatin. Mouse ES cells are derived from the inner cell mass (ICM) of blastocyst stage embryos prior to X-inactivation, and cultured murine ES cells initiate this process only upon differentiation. Less is known about human X-inactivation during early development. To identify a human ES cell model for X-inactivation and study differences in the epigenetic state of hESC lines, we investigated X-inactivation in all growth competent, karyotypically normal, NIH approved, female hESC lines and several sublines. In the vast majority of undifferentiated cultures of nine lines examined, essentially all cells exhibit hallmarks of X-inactivation. However, subcultures of any hESC line can vary in X-inactivation status, comprising distinct sublines. Importantly, we identified rare sublines that have not yet inactivated Xi and retain competence to undergo X-inactivation upon differentiation. Other sublines exhibit defects in counting or maintenance of XIST expression on Xi. The few hESC sublines identified that have not yet inactivated Xi may reflect the earlier epigenetic state of the human ICM and represent the most promising source of NIH hESC for study of human X-inactivation. The many epigenetic anomalies seen indicate that maintenance of fully unspecialized cells, which have not formed Xi facultative heterochromatin, is a delicate epigenetic balance difficult to maintain in culture. PMID:18340642

  7. Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling

    PubMed Central

    Hay, David C.; Fletcher, Judy; Payne, Catherine; Terrace, John D.; Gallagher, Ronald C. J.; Snoeys, Jan; Black, James R.; Wojtacha, Davina; Samuel, Kay; Hannoun, Zara; Pryde, Anne; Filippi, Celine; Currie, Ian S.; Forbes, Stuart J.; Ross, James A.; Newsome, Philip N.; Iredale, John P.

    2008-01-01

    Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function. PMID:18719101

  8. Nanofibrous substrates support colony formation and maintain stemness of human embryonic stem cells

    PubMed Central

    Gauthaman, Kalamegam; Venugopal, Jayarama Reddy; Yee, Fong Chui; Peh, Gary Swee Lim; Ramakrishna, Seeram; Bongso, Ariff

    2009-01-01

    Inadequate cell numbers in culture is one of the hurdles currently delaying the application of human embryonic stem cells (hESCs) for transplantation therapy. Nanofibrous scaffolds have been effectively used to expand and differentiate non-colony forming multipotent mesenchymal stem cells (MSC) for the repair of tissues or organs. In the present study, we evaluated the influence of nanofibrous scaffolds for hESC proliferation, increase in colony formation, self-renewal properties, undifferentiation and retention of ‘stemness’. Polycaprolactone/collagen (PCL/collagen) and PCL/gelatin nanofibrous scaffolds were fabricated using electrospinning technology. The hESCs were seeded on the nanofibrous scaffolds in the presence or absence of mitomycin-C treated mouse embryonic fibroblasts (MEFs). The hESCs grown on both scaffolds in the presence of the MEFs produced an increase in cell growth of 47.58% (P≤ 0.006) and 40.18% (P≤ 0.005), respectively, over conventional controls of hESCs on MEFs alone. The hESC colonies were also larger in diameter on the scaffolds compared to controls (PCL/collagen, 156.25 ± 7 μM and PCL/gelatin, 135.42 ± 5 μM). Immunohistochemistry of the hESCs grown on the nanofibrous scaffolds with MEFs, demonstrated positive staining for the various stemness-related markers (octamer 4 [OCT-4], tumour rejection antigen-1–60, GCTM-2 and TG-30), and semi-quantitative RT-PCR for the pluripotent stemness genomic markers (NANOG, SOX-2, OCT-4) showed that they were also highly expressed. Continued successful propagation of hESC colonies from nanofibrous scaffolds back to conventional culture on MEFs was also possible. Nanofibrous scaffolds support hESC expansion in an undifferentiated state with retention of stemness characteristics thus having tremendous potential in scaling up cell numbers for transplantation therapy. PMID:19228268

  9. Platinum blue staining of cells grown in electrospun scaffolds.

    PubMed

    Yusuf, Mohammed; Millas, Ana Luiza G; Estandarte, Ana Katrina C; Bhella, Gurdeep K; McKean, Robert; Bittencourt, Edison; Robinson, Ian K

    2014-01-01

    Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

  10. OM-VPE grown materials for high efficiency solar cells

    NASA Technical Reports Server (NTRS)

    Saxena, R.; Cooper, B., III; Ludowise, M.; Borden, P.; Gregory, P.

    1980-01-01

    Organometallic sources are available for all the III-V elements and a variety of dopants; thus it is possible to use the technique to grow a wide variety of semiconductor compounds. AlGaAsSb and AlGaInAs alloys for multijunction monolithic solar cells were grown by OM-VPE. While the effort concentrated on terrestrial applications, the success of OM-VPE grown GaAs/AlGaAs concentrator solar cells (23% at 400 suns) demonstrates that OM-VPE is suitable for growing high efficiency solar cells in large quantities for space applications. In addition, OM-VPE offers the potential for substantial cost reduction of photovoltaic devices with scale up and automation and due to high process yield from reproducible, uniform epitaxial growths with excellent surface morphology.

  11. Human RPE Stem Cells Grown into Polarized RPE Monolayers on a Polyester Matrix Are Maintained after Grafting into Rabbit Subretinal Space

    PubMed Central

    Stanzel, Boris V.; Liu, Zengping; Somboonthanakij, Sudawadee; Wongsawad, Warapat; Brinken, Ralf; Eter, Nicole; Corneo, Barbara; Holz, Frank G.; Temple, Sally; Stern, Jeffrey H.; Blenkinsop, Timothy A.

    2014-01-01

    Summary Transplantation of the retinal pigment epithelium (RPE) is being developed as a cell-replacement therapy for age-related macular degeneration. Human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC)-derived RPE are currently translating toward clinic. We introduce the adult human RPE stem cell (hRPESC) as an alternative RPE source. Polarized monolayers of adult hRPESC-derived RPE grown on polyester (PET) membranes had near-native characteristics. Trephined pieces of RPE monolayers on PET were transplanted subretinally in the rabbit, a large-eyed animal model. After 4 days, retinal edema was observed above the implant, detected by spectral domain optical coherence tomography (SD-OCT) and fundoscopy. At 1 week, retinal atrophy overlying the fetal or adult transplant was observed, remaining stable thereafter. Histology obtained 4 weeks after implantation confirmed a continuous polarized human RPE monolayer on PET. Taken together, the xeno-RPE survived with retained characteristics in the subretinal space. These experiments support that adult hRPESC-derived RPE are a potential source for transplantation therapies. PMID:24511471

  12. Coupled Global and Targeted Proteomics of Human Embryonic Stem Cells during Induced Differentiation*S⃞

    PubMed Central

    Yocum, Anastasia K.; Gratsch, Theresa E.; Leff, Nancy; Strahler, John R.; Hunter, Christie L.; Walker, Angela K.; Michailidis, George; Omenn, Gilbert S.; O'Shea, K. Sue; Andrews, Philip C.

    2008-01-01

    Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomics studies. The current investigation avoided the use of MEFs or MEF-conditioned medium for hESC culture, allowing global proteomics analysis without these confounding conditions, and elucidated neural cell-specific signaling pathways involved in noggin-induced hESC differentiation. Based on these analyses, we propose the following early markers of hESC neural differentiation: collapsin response mediator proteins 2 and 4 and the nuclear autoantigenic sperm protein as a marker of pluripotent hESCs. We then developed a directed mass spectrometry assay using multiple reaction monitoring (MRM) to identify and quantify these markers and in addition the epidermal ectoderm marker cytokeratin-8. Analysis of global proteomics, quantitative RT-PCR, and MRM data led to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show that targeted MRM analysis on non-redundant peptides provides more exact quantification of homologous proteins. This study describes the facile transition from discovery proteomics to targeted MRM analysis and allowed us to identify and verify several potential biomarkers for hESCs during noggin-induced neural and BMP4-induced epidermal ectoderm differentiation. PMID:18304949

  13. Photosynthetic Characteristics of Photoautotrophically Grown Tobacco Callus Cells 1

    PubMed Central

    Berlyn, Mary B.; Zelitch, Israel; Beaudette, Pamela D.

    1978-01-01

    Haploid callus cells of tobacco (Nicotiana tabacum) were grown photoautotrophically on a solid agar medium in the absence of sucrose in Petri plates in an atmosphere of 1% or 3% CO2 in air. The averages of dry weight increases for four to five consecutive passages were 2.3- to 3.6-fold per 3-week passage for different subclones. Photosynthetic 14CO2 assimilation was maximum at about 1% CO2 with half-maximal rates obtained at 0.2% CO2. At saturating CO2 concentration the average rate of CO2 fixation was about 5 μmole per gram fresh weight per hour or about 125 μmole per mg of chlorophyll per hour. The existence of an active photorespiratory system in these tissues was established in a number of independent ways. The photosynthetic rate in 0.18% CO2 was inhibited 38 to 50% in 100% O2 compared with 21% O2. Glycolate accumulated at a constant rate in the presence of 5 mm α-hydroxy-2-pyridinemethanesulfonic acid for 20 minutes in light. This rate was rapid relative to the photosynthetic rate. Glycolate synthesis was three times faster in autotrophic than in heterotrophic cells. [1-14C]Glycolate was rapidly metabolized and the products included 14CO2, [14C]glycine, and [14C]serine, thus demonstrating an active glycolate pathway. Photorespiration was demonstrated directly by measurement of an O2-dependent release of 14CO2 in the light from callus that fixed 14CO2 for about 22 hours. Autotrophic growth in 60% O2 and 0.03% CO2 was slowed and ceased entirely after two or three passages, while heterotrophic growth was unaffected by 60% O2 in the atmosphere. The method of growing autotrophic callus which has an active photorespiratory system should facilitate the selection and analysis of photosynthetic mutants in which photorespiration is regulated. PMID:16660346

  14. Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells

    PubMed Central

    Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

    2015-01-01

    Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice. PMID:26091287

  15. Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells.

    PubMed

    Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

    2015-01-01

    Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

  16. Pro-angiogenic Cell Colonies Grown In Vitro from Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Mavromatis, Kreton; Sutcliffe, Diane; Joseph, Giji; Alexander, R. Wayne; Waller, Edmund K.; Quyyumi, Arshed A.; Taylor, W. Robert

    2014-01-01

    Although multiple culture assays have been designed to identify “endothelial progenitor cells” (EPCs), the phenotype of cells grown in culture often remains undefined. We sought to define and characterize the pro-angiogenic cell population within human peripheral blood mononuclear cells. Mononuclear cells were isolated from peripheral blood and grown under angiogenic conditions for 7 days. Formed colonies (CFU-As) were identified and analyzed for proliferation, mRNA and surface antigen expression, tube-forming ability and chromosomal content. Colonies were composed of a heterogeneous group of cells expressing the leukocyte antigens CD45, CD14, and CD3, as well as the endothelial proteins vascular endothelial (VE) cadherin, von Willebrand's Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS). Colony cells expressed increased levels of pro-angiogenic growth factors, and they formed tubes in Matrigel. In comparison with colonies from the CFU-Hill assay, our assay resulted in a greater number of colonies (19±9 vs. 13±7; p<0.0001) with a substantial number of cells expressing an endothelial phenotype (20.2±7.4% vs. 2.2±1.2% expressing eNOS, p=0006). Chromosomal analysis indicated the colony cells were bone marrow-derived. We, therefore, describe a colony forming unit assay that measures bone marrow-derived circulating mononuclear cells with the capacity to proliferate and mature into proangiogenic leukocytic and endothelial-like cells. This assay, therefore, reflects circulating, bone marrow-derived pro-angiogenic activity. PMID:22904201

  17. Separation and Ultrastructure of Proplastids from Dark-grown Euglena Cells

    PubMed Central

    Ophir, Ilana; Ben-Shaul, Yehuda

    1973-01-01

    A procedure for the separation of proplastids free of mitochondria from dark-grown Euglena cells has been developed. A fraction enriched in proplastids was used for freeze-etching study of proplastid structure. The prolamellar body in freeze-etched replicas appeared sponge-like, with thylakoids, often vesicular, emerging from it. The prolamellar body and the thylakoids were covered by particles of about 100Å in diameter. No larger particles, typical of light-grown chloroplasts, were observed. Images PMID:16658476

  18. Separation and Ultrastructure of Proplastids from Dark-grown Euglena Cells.

    PubMed

    Ophir, I; Ben-Shaul, Y

    1973-06-01

    A procedure for the separation of proplastids free of mitochondria from dark-grown Euglena cells has been developed. A fraction enriched in proplastids was used for freeze-etching study of proplastid structure. The prolamellar body in freeze-etched replicas appeared sponge-like, with thylakoids, often vesicular, emerging from it. The prolamellar body and the thylakoids were covered by particles of about 100A in diameter. No larger particles, typical of light-grown chloroplasts, were observed.

  19. Phenotypic expression of marrow cells when grown on various substrata.

    PubMed

    Fried, A; Shamay, A; Wientroub, S; Benayahu, D

    1996-05-01

    Our aim was to study the role of various extracellular matrices (ECM) on growth and differentiation of marrow stromal cells in vitro. Morphology changes, gene expression, and enzymatic activities were monitored in stromal osteoblastic MBA-15 and adipocytic 14F1.1 cells. These stromal cells were plated on dishes precoated with different substrata, such as matrigel (basement membrane), collagen type I, and endothelial ECM, and compared with cells plated on protein-free dishes. Striking morphological differences were observed when the cells grew on these different substrata. Changes in cell shape and growth also led to differential mRNA expression and enzymatic activities. When MBA-15 cells were plated on collagen, there was a decrease in mRNA for alkaline phosphatase (ALK-P), osteopontin (OP), and osteonectin (ON), and an increase in mRNA for procollagen (I). A differential effect was noted on 14F1.1 cells, the mRNA for ALK-P increased, the expressions of OP and ON lowered, and no expression for procollagen (I) was monitored. MBA-15 cells cultured on matrigel had decreased mRNA for ALK-P and OP, while they had increased ON mRNA expression and remained unchanged for procollagen I. No change in mRNA expression by 14F1.1 cells was monitored when cultured on matrigel. Functional enzymatic activities of ALK-P markedly decreased in MBA-15 cells cultured on various substrata, and increased or were unchanged in 14F1.1 cells. An additional enzyme, neutral endopeptidase (CD10/NEP), altered differentially in both cell types; this enzymatic activity increased or was unchanged when cells were cultured on these matrices. The results indicate a specific role for different ECM on various stromal cell types and their function. PMID:9173088

  20. Human Lung Cancer Cells Grown on Acellular Rat Lung Matrix Create Perfusable Tumor Nodules

    PubMed Central

    Mishra, Dhruva K.; Thrall, Michael J.; Baird, Brandi N.; Ott, Harald C.; Blackmon, Shanda H.; Kurie, Jonathan M.; Kim, Min P.

    2015-01-01

    Background Extracellular matrix allows lung cancer to form its shape and grow. Recent studies on organ reengineering for orthotopic transplantation have provided a new avenue for isolating purified native matrix to use for growing cells. Whether human lung cancer cells grown in a decellularized rat lung matrix would create perfusable human lung cancer nodules was tested. Methods Rat lungs were harvested and native cells were removed using sodium dodecyl sulfate and Triton X-100 in a decellularization chamber to create a decellularized rat lung matrix. Human A549, H460, or H1299 lung cancer cells were placed into the decellularized rat lung matrix and grown in a customized bioreactor with perfusion of oxygenated media for 7 to 14 days. Results Decellularized rat lung matrix showed preservation of matrix architecture devoid of all rat cells. All three human lung cancer cell lines grown in the bioreactor developed tumor nodules with intact vasculature. Moreover, the lung cancer cells developed a pattern of growth similar to the original human lung cancer. Conclusions Overall, this study shows that human lung cancer cells form perfusable tumor nodules in a customized bioreactor on a decellularized rat lung matrix created by a customized decellularization chamber. The lung cancer cells grown in the matrix had features similar to the original human lung cancer. This ex vivo model can be used potentially to gain a deeper understanding of the biologic processes involved in human lung cancer. PMID:22385822

  1. RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells.

    PubMed

    Sigal, Nadejda; Pasechnek, Anna; Herskovits, Anat A

    2016-01-01

    Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells (1-4). The protocol can be easily modified to study other bacterial pathogens and cell types. PMID:27341521

  2. Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate.

    PubMed

    Pennington, Britney O; Clegg, Dennis O; Melkoumian, Zara K; Hikita, Sherry T

    2015-02-01

    Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies. PMID:25593208

  3. Primary human bronchial epithelial cells grown from explants.

    PubMed

    Yaghi, Asma; Zaman, Aisha; Dolovich, Myrna

    2010-01-01

    Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and < or =1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm(3) pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 microg/ml), fibronectin (10 microg/ml), and BSA (10 microg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37 degrees C in 5% CO(2) humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly

  4. Endocytic activity of Sertoli cells grown in bicameral culture chambers

    SciTech Connect

    Dai, R.X.; Djakiew, D.; Dym, M.

    1987-07-01

    Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding /sup 125/I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived.

  5. Localized decrease of {beta}-catenin contributes to the differentiation of human embryonic stem cells

    SciTech Connect

    Lam, Hayley; Patel, Shyam; Wong, Janelle; Chu, Julia; Li, Adrian; Li, Song

    2008-08-08

    Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, {beta}-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of {beta}-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down {beta}-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of {beta}-catenin contributes to the spatial pattern of differentiation in hESC colonies.

  6. Changes in Escherichia coli cells starved in seawater or grown in seawater-wastewater mixtures.

    PubMed Central

    Munro, P M; Gauthier, M J; Laumond, F M

    1987-01-01

    Some metabolic modifications of Escherichia coli cells during starvation in seawater were studied in laboratory microcosms. The apparent die-off of this bacterium under such conditions, as observed by comparing the enumeration of CFU in conventional freshwater media and direct epifluorescence counts, was partially prevented when cells were previously grown in salted organic medium or on seawater-wastewater agar. beta-Galactosidase activity of starved cells disappeared gradually with time, even though some other enzymatic activities, such as that of alkaline phosphatase, increased. Moreover, some modifications of sensitivity to antibiotics, heavy metals, and bacteriophages in seawater- and wastewater-grown cells suggested that the cells undergo structural changes under natural marine conditions. These results provide additional experimental data indicating the possible active adaptation of E. coli cells to seawater. PMID:3116927

  7. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells

    PubMed Central

    Danevčič, Tjaša; Borić Vezjak, Maja; Tabor, Maja; Zorec, Maša; Stopar, David

    2016-01-01

    Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent. PMID:26858704

  8. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells.

    PubMed

    Danevčič, Tjaša; Borić Vezjak, Maja; Tabor, Maja; Zorec, Maša; Stopar, David

    2016-01-01

    Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent.

  9. Mechanics Regulates Fate Decisions of Human Embryonic Stem Cells

    PubMed Central

    Sun, Yubing; Villa-Diaz, Luis G.; Lam, Raymond H. W.; Chen, Weiqiang; Krebsbach, Paul H.; Fu, Jianping

    2012-01-01

    Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function. PMID:22615930

  10. Ethanol Negatively Regulates Hepatic Differentiation of hESC by Inhibition of the MAPK/ERK Signaling Pathway In Vitro

    PubMed Central

    Ma, Xiaocui; Tschudy-Seney, Benjamin; Chen, Jiamei; Magner, Nataly L.; Revzin, Alexander; Nolta, Jan A.; Zern, Mark A.; Duan, Yuyou

    2014-01-01

    Background Alcohol insult triggers complex events in the liver, promoting fibrogenic/inflammatory signals and in more advanced cases, aberrant matrix deposition. It is well accepted that the regenerative capacity of the adult liver is impaired during alcohol injury. The liver progenitor/stem cells have been shown to play an important role in liver regeneration -in response to various chronic injuries; however, the effects of alcohol on stem cell differentiation in the liver are not well understood. Methods We employed hepatic progenitor cells derived from hESCs to study the impact of ethanol on hepatocyte differentiation by exposure of these progenitor cells to ethanol during hepatocyte differentiation. Results We found that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitor cells in a dose-dependent manner. There was also a moderate cell cycle arrest at G1/S checkpoint in the ethanol treated cells, which is associated with a reduced level of cyclin D1 in these cells. Ethanol treatment specifically inhibited the activation of the ERK but not JNK nor the p38 MAP signaling pathway. At the same time, the WNT signaling pathway was also reduced in the cells exposed to ethanol. Upon evaluating the effects of the inhibitors of these two signaling pathways, we determined that the Erk inhibitor replicated the effects of ethanol on the hepatocyte differentiation and attenuated the WNT/β-catenin signaling, however, inhibitors of WNT only partially replicated the effects of ethanol on the hepatocyte differentiation. Conclusion Our results demonstrated that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitors through inhibiting the MAPK/ERK signaling pathway, and subsequently attenuating the WNT signaling pathway. Thus, our finding provides a novel insight into the mechanism by which alcohol regulates cell fate selection of hESC-derived hepatic progenitor cells, and the identified pathways may provide

  11. Plastid distribution in columella cells of a starchless Arabidopsis mutant grown in microgravity

    NASA Technical Reports Server (NTRS)

    Hilaire, E.; Paulsen, A. Q.; Brown, C. S.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1997-01-01

    Wild-type and starchless Arabidopsis thaliana mutant seedlings (TC7) were grown and fixed in the microgravity environment of a U.S. Space Shuttle spaceflight. Computer image analysis of longitudinal sections from columella cells suggest a different plastid positioning mechanism for mutant and wild-type in the absence of gravity.

  12. High-Fidelity Reprogrammed Human IPSCs Have a High Efficacy of DNA Repair and Resemble hESCs in Their MYC Transcriptional Signature.

    PubMed

    Nagaria, Pratik K; Robert, Carine; Park, Tea Soon; Huo, Jeffrey S; Zambidis, Elias T; Rassool, Feyruz V

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) are reprogrammed from adult or progenitor somatic cells and must make substantial adaptations to ensure genomic stability in order to become "embryonic stem cell- (ESC-) like." The DNA damage response (DDR) is critical for maintenance of such genomic integrity. Herein, we determined whether cell of origin and reprogramming method influence the DDR of hiPSCs. We demonstrate that hiPSCs derived from cord blood (CB) myeloid progenitors (i.e., CB-iPSC) via an efficient high-fidelity stromal-activated (sa) method closely resembled hESCs in DNA repair gene expression signature and irradiation-induced DDR, relative to hiPSCs generated from CB or fibroblasts via standard methods. Furthermore, sa-CB-iPSCs also more closely resembled hESCs in accuracy of nonhomologous end joining (NHEJ), DNA double-strand break (DSB) repair, and C-MYC transcriptional signatures, relative to standard hiPSCs. Our data suggests that hiPSCs derived via more efficient reprogramming methods possess more hESC-like activated MYC signatures and DDR signaling. Thus, an authentic MYC molecular signature may serve as an important biomarker in characterizing the genomic integrity in hiPSCs. PMID:27688775

  13. High-Fidelity Reprogrammed Human IPSCs Have a High Efficacy of DNA Repair and Resemble hESCs in Their MYC Transcriptional Signature

    PubMed Central

    Park, Tea Soon; Zambidis, Elias T.

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) are reprogrammed from adult or progenitor somatic cells and must make substantial adaptations to ensure genomic stability in order to become “embryonic stem cell- (ESC-) like.” The DNA damage response (DDR) is critical for maintenance of such genomic integrity. Herein, we determined whether cell of origin and reprogramming method influence the DDR of hiPSCs. We demonstrate that hiPSCs derived from cord blood (CB) myeloid progenitors (i.e., CB-iPSC) via an efficient high-fidelity stromal-activated (sa) method closely resembled hESCs in DNA repair gene expression signature and irradiation-induced DDR, relative to hiPSCs generated from CB or fibroblasts via standard methods. Furthermore, sa-CB-iPSCs also more closely resembled hESCs in accuracy of nonhomologous end joining (NHEJ), DNA double-strand break (DSB) repair, and C-MYC transcriptional signatures, relative to standard hiPSCs. Our data suggests that hiPSCs derived via more efficient reprogramming methods possess more hESC-like activated MYC signatures and DDR signaling. Thus, an authentic MYC molecular signature may serve as an important biomarker in characterizing the genomic integrity in hiPSCs.

  14. High-Fidelity Reprogrammed Human IPSCs Have a High Efficacy of DNA Repair and Resemble hESCs in Their MYC Transcriptional Signature

    PubMed Central

    Park, Tea Soon; Zambidis, Elias T.

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) are reprogrammed from adult or progenitor somatic cells and must make substantial adaptations to ensure genomic stability in order to become “embryonic stem cell- (ESC-) like.” The DNA damage response (DDR) is critical for maintenance of such genomic integrity. Herein, we determined whether cell of origin and reprogramming method influence the DDR of hiPSCs. We demonstrate that hiPSCs derived from cord blood (CB) myeloid progenitors (i.e., CB-iPSC) via an efficient high-fidelity stromal-activated (sa) method closely resembled hESCs in DNA repair gene expression signature and irradiation-induced DDR, relative to hiPSCs generated from CB or fibroblasts via standard methods. Furthermore, sa-CB-iPSCs also more closely resembled hESCs in accuracy of nonhomologous end joining (NHEJ), DNA double-strand break (DSB) repair, and C-MYC transcriptional signatures, relative to standard hiPSCs. Our data suggests that hiPSCs derived via more efficient reprogramming methods possess more hESC-like activated MYC signatures and DDR signaling. Thus, an authentic MYC molecular signature may serve as an important biomarker in characterizing the genomic integrity in hiPSCs. PMID:27688775

  15. Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media.

    PubMed

    McIntyre, D A; Harlander, S K

    1989-10-01

    The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample. PMID:2513778

  16. X-ray responses of human colon tumor cells grown in artificial capillary culture

    SciTech Connect

    Nakazawa, M.; Leith, J.T.; Glicksman, A.S.

    1984-06-01

    Clone A human colon adenocarcinoma cells were grown in three-dimensional artificial capillary culture (ACC) to determine responses of capillaries treated 3 weeks after tumor cell inoculation with a specific, easily quantifiable cytotoxic agent, ionizing radiation. Changes in extracapillary space (ECS) fluid concentrations of lactate dehydrogenase (LDH) and aspartate aminotransferase (GOT) and the utilization of glucose in circulating medium were monitored after a supralethal radiation dose (90 Gy) of X-rays. Immediately after irradiation, increased levels of LDH and GOT were found that reached maximum levels about four to five times those found in nonirradiated control capillaries at 10-13 days post irradiation and then declined. Patterns of enzyme production appeared to correlate with the numbers of nonviable tumor cells collected from the ECS of the artificial capillaries. In contrast, glucose utilization showed little correlation with either enzyme concentration or dead cell production. In other studies, tumor cells were removed from unirradiated capillaries by trypsinization and used to obtain complete survival curves after graded doses of X-radiation. The dose-response curves obtained indicate that clone A colon tumor cells grown in ACC show a marked decrease in their ability to accumulate sublethal radiation injury as compared to responses of these cells growing exponentially in asynchronous monolayer cultures, to synchronized mid-G1 tumor cells, or to tumor cells in stationary growth phase. These data suggest that ACC is a potentially useful model to study the effects of cytotoxic agents on human tumor cells.

  17. Homojunction GaAs solar cells grown by close space vapor transport

    SciTech Connect

    Boucher, Jason W.; Ritenour, Andrew J.; Greenaway, Ann L.; Aloni, Shaul; Boettcher, Shannon W.

    2014-06-08

    We report on the first pn junction solar cells grown by homoepitaxy of GaAs using close space vapor transport (CSVT). Cells were grown both on commercial wafer substrates and on a CSVT absorber film, and had efficiencies reaching 8.1%, open circuit voltages reaching 909 mV, and internal quantum efficiency of 90%. The performance of these cells is partly limited by the electron diffusion lengths in the wafer substrates, as evidenced by the improved peak internal quantum efficiency in devices fabricated on a CSVT absorber film. Unoptimized highly-doped n-type emitters also limit the photocurrent, indicating that thinner emitters with reduced doping, and ultimately wider band gap window or surface passivation layers, are required to increase the efficiency.

  18. Effects of method of detachment on electrophoretic mobility of mammalian cells grown in monolayer culture

    NASA Technical Reports Server (NTRS)

    Plank, L. D.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    A variety of proteolytic and micolytic enzumes, mechanical procedures, and changes in the ionic environment, especially Ca chelation, are used for dispersal of monolayer grown cells. If either chelating agents or mechanical dispersion are used alone, the cell yield is often low and suspensions of single cells are difficult to obtain. Confluent monolayers treated with EDTA tend to be released from their surfaces in sheets, and clumps of cells remain even after further incubation in EDTA. Crude trypsin is the most popular dispersal agent and is known to contain a variety of contaminating enzymes which contribute to the dispersal of cells. A variety of cell injuries resulting from the activity of proteolytic enzymes are reported. It is shown that crystalline trypsin is least harmful to cell integrity as judged by trypan blue uptake.

  19. Photoelectrochemical cell using dye sensitized zinc oxide nanowires grown on carbon fibers

    NASA Astrophysics Data System (ADS)

    Unalan, Husnu Emrah; Wei, Di; Suzuki, Kenichi; Dalal, Sharvari; Hiralal, Pritesh; Matsumoto, Hidetoshi; Imaizumi, Shinji; Minagawa, Mie; Tanioka, Akihiko; Flewitt, Andrew J.; Milne, William I.; Amaratunga, Gehan A. J.

    2008-09-01

    Zinc oxide (ZnO) nanowires (NWs) grown on carbon fibers using a vapor transport and condensation approach are used as the cathode of a photoelectrochemical cell. The carbon fibers were obtained by electrospray deposition and take the form of a flexible carbon fabric. The ZnO NW on carbon fiber anode is combined with a "black dye" photoabsorber, an electrolyte, and a platinum (Pt) counterelectrode to complete the cell. The results show that ZnO NW and carbon fibers can be used for photoinduced charge separation/charge transport and current collection, respectively, in a photoelectrochemical cell.

  20. Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

    PubMed Central

    Burke, David; Keegstra, Kenneth

    1979-01-01

    Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways. PMID:430605

  1. Longevity of U cells of differentiated yeast colonies grown on respiratory medium depends on active glycolysis.

    PubMed

    Čáp, Michal; Váchová, Libuše; Palková, Zdena

    2015-01-01

    Colonies of Saccharomyces cerevisiae laboratory strains pass through specific developmental phases when growing on solid respiratory medium. During entry into the so-called alkali phase, in which ammonia signaling is initiated, 2 prominent cell types are formed within the colonies: U cells in upper colony regions, which have a longevity phenotype and activate the expression of a large number of metabolic genes, and L cells in lower regions, which die more quickly and exhibit a starvation phenotype. Here, we performed a detailed analysis of the activities of enzymes of central carbon metabolism in lysates of both cell types and determined several fermentation end products, showing that previously reported expression differences are reflected in the different enzymatic capabilities of each cell type. Hence, U cells, despite being grown on respiratory medium, behave as fermenting cells, whereas L cells rely on respiratory metabolism and possess active gluconeogenesis. Using a spectrum of different inhibitors, we showed that glycolysis is essential for the formation, and particularly, the survival of U cells. We also showed that β-1,3-glucans that are released from the cell walls of L cells are the most likely source of carbohydrates for U cells.

  2. Epitaxial Crystal Silicon Absorber Layers and Solar Cells Grown at 1.8 Microns per Minute

    SciTech Connect

    Bobela, D. C.; Teplin, C. W.; Young, D. L.; Branz, H. M.; Stradins, P.

    2011-01-01

    We have grown device-quality epitaxial silicon thin films at growth rates up to 1.85 {micro}m/min, using hot-wire chemical vapor deposition from silane, at substrate temperatures below 750 C. At these rates, which are more than 30 times faster than those used by the amorphous and nanocrystalline Si industry, capital costs for large-scale solar cell production would be dramatically reduced, even for cell absorber layers up to 10 {micro}m thick. We achieved high growth rates by optimizing the three key parameters: silane flow, depletion, and filament geometry, based on our model developed earlier. Hydrogen coverage of the filament surface likely limits silane decomposition and growth rate at high system pressures. No considerable deterioration in PV device performance is observed when grown at high rate, provided that the epitaxial growth is initiated at low rate. A simple mesa device structure (wafer/epi Si/a-Si(i)/a-Si:H(p)/ITO) with a 2.3 {micro}m thick epitaxial silicon absorber layer was grown at 0.7 {micro}m/min. The finished device had an open-circuit voltage of 0.424 V without hydrogenation treatment.

  3. Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis

    PubMed Central

    ABUNA, Rodrigo Paolo Flores; STRINGHETTA-GARCIA, Camila Tami; FIORI, Leonardo Pimentel; DORNELLES, Rita Cassia Menegati; ROSA, Adalberto Luiz; BELOTI, Marcio Mateus

    2016-01-01

    ABSTRACT Aging negatively affects bone/titanium implant interactions. Our hypothesis is that the unbalance between osteogenesis and adipogenesis induced by aging may be involved in this phenomenon. Objective We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. Material and Methods Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. Results Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. Conclusions These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population. PMID:27556209

  4. Epitaxially grown polycrystalline silicon thin-film solar cells on solid-phase crystallised seed layers

    NASA Astrophysics Data System (ADS)

    Li, Wei; Varlamov, Sergey; Xue, Chaowei

    2014-09-01

    This paper presents the fabrication of poly-Si thin film solar cells on glass substrates using seed layer approach. The solid-phase crystallised P-doped seed layer is not only used as the crystalline template for the epitaxial growth but also as the emitter for the solar cell structure. This paper investigates two important factors, surface cleaning and intragrain defects elimination for the seed layer, which can greatly influence the epitaxial grown solar cell performance. Shorter incubation and crystallisation time is observed using a simplified RCA cleaning than the other two wet chemical cleaning methods, indicating a cleaner seed layer surface is achieved. Cross sectional transmission microscope images confirm a crystallographic transferal of information from the simplified RCA cleaned seed layer into the epi-layer. RTA for the SPC seed layer can effectively eliminate the intragrain defects in the seed layer and improve structural quality of both of the seed layer and the epi-layer. Consequently, epitaxial grown poly-Si solar cell on the RTA treated seed layer shows better solar cell efficiency, Voc and Jsc than the one on the seed layer without RTA treatment.

  5. DNA content and differentiation of root apical cells of Brassica rapa plants grown in microgravity.

    PubMed

    Kordyum, E L; Martin, G I; Zaslavsky, V A; Jiao, S; Hilaire, E; Guikema, J A

    1999-07-01

    Root cap is proposed to be a graviperceptive tissue in the plant root, and it is composed of several cell types. One such cell type, the columella cells, are thought to initiate the gravity-induced signal transduction cascade, and these cells arise from the activity of the meristematic zone of the root cap. There is, in fact, a continuum of cells in the central column of the root cap representing the meristematic cells, developing columella cells, mature cells, and those that will soon be sloughed off into the soil. In order to study the functional roles of the root cap cells in gravity-sensing, we compared the ultrastructural organization, differentiation, and DNA content in the meristematic, elongating, and differentiating cells of root tips in Brassica rapa plants grown in space microgravity and at 1g. The experiments were also designed to determine the reactions of root cap cells in both main roots (in which the original root cap was present in an embryonic form within the seed) and lateral roots (in which the root cap formed completely in space after seed germination on orbit) to the space microgravity. This study (ROOTS) was performed in collaboration with the B-PAC experiment on the Space shuttle "Columbia" mission STS-87 (Collaborative US/Ukrainian Experiment (CUE) during November 19-December 5, 1997.

  6. Chondrogenesis in aggregates of embryonic limb cells grown in a rotating wall vessel

    NASA Astrophysics Data System (ADS)

    Duke, J.; Daane, E.; Arizpe, J.; Montufar-Solis, D.

    Previous studies in this lab have shown that chondrogenesis is affected in growth plates of rats exposed to microgravity, and in micromass cultures of embryonic limb mesenchyme differentiating in space. In order to provide a three dimensional aspect not seen in the micromass system, and a tissue homogeneity not possible with explants of limb or limb elements, and to alleviate certain difficulties regarding crew time and stowage, we began culturing embryonic limb cells in Rotating Wall Vessels (RWV). First, these cells were attached to beads, and grown for up to 65 days in a type of RWV known as STLV at the Johnson Space Center. During this time, the cells and beads aggregated and the aggregates continued to increase in size, and differentiated into Alcian blue staining chondrocytes. Because our intent was to use these aggregates for implanting into bony defects in addition to their use in studies of chondrogenic regulation at 1g and mug, aggregates of these cells without beads were grown in the commercially available version of the STLV, and their ability to ossify when subcutaneously implanted assessed.

  7. Influence of chronological aging on the survival and nucleotide content of Saccharomyces cerevisiae cells grown in different conditions: occurrence of a high concentration of UDP-N-acetylglucosamine in stationary cells grown in 2% glucose.

    PubMed

    Osório, Hugo; Silles, Eduardo; Maia, Rita; Peleteiro, Bárbara; Moradas-Ferreira, Pedro; Günther Sillero, María A; Sillero, Antonio

    2005-02-01

    Saccharomyces cerevisiae cells (strain W303) grown in a minimal medium (containing 2% or 0.1% glucose) until exponential or stationary phase, were subjected to chronological aging in water, and yeast viability and nucleotide content were analyzed along several days of nutrient starvation. Cells collected in exponential phase (whether grown in the presence of 0.1% or 2% glucose) were viable up to five days and thereafter the viability decreased linearly with a half-survival rate of around eight days. ATP and other nucleoside triphosphates decreased similarly in both cases. Cells collected in stationary phase, and transferred to water, behaved differently whether grown in 0.1% or in 2% glucose, with a half-survival life of around nine and 28 days respectively. A double mutant in glycogen synthase (gsy1delta gsy2delta) and its isogenic wild-type strain, grown to stationary phase in 2% glucose, presented a similar half-survival life of around eight days. The W303 cells grown to stationary phase in the presence of 2% glucose showed a 7-fold increase of UDP-N-acetylglucosamine (UDP-GlcNAc) as compared with the level present in the cells grown in any of the other three metabolic situations. The nature of UDP-GlcNAc was established by MALDI-TOF ionization analysis. It is also worth noting that the rate of decay of NAD+ was lower than that of ATP in any of the situations here considered.

  8. Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture.

    PubMed

    Chaplen, F W; Fahl, W E; Cameron, D C

    1996-07-01

    Methylglyoxal is present at low levels in most cells as a by-product of glycolysis and a product of lipid and amino acid catabolism. The most widely accepted method for measurement of methylglyoxal involves the derivatization of methylglyoxal with 1,2-diaminobenzene derivatives, such as o-phenylenediamine, followed by quantification of the resulting quinoxaline with high-performance liquid chromatography (HPLC). Here we describe the modification of this procedure for the measurement of free intra- and extracellular methylglyoxal in animal cells grown in culture. Cell harvest and sample volume measurement techniques were developed. Solid-phase extraction prior to methylglyoxal derivatization reduced interferences unique to cell culture, such as the phenol red indicator dye used in most cell culture media, and extended the useful life of the HPLC column. In addition, this extraction step significantly lessened the interference represented by oxidative degradation of nucleic acids to methylglyoxal by perchloric acid under assay conditions. The concentration of free intracellular methylglyoxal in Chinese hamster ovary (CHO) cells grown in culture ranged from 0.7 +/- 0.3 microM (mean +/- 2 standard deviations; n = 4) to 1.2 +/- 0.3 microM (mean +/- 2 standard deviations; n = 7). The concentration of free extracellular methylglyoxal in the growth medium was 0.07 +/- 0.02 microM (mean +/- 2 standard deviations; n = 4), severalfold less than that found inside the cell. A possible explanation for the difference between measured free intracellular and extracellular methylglyoxal levels is that the assay for free intracellular methylglyoxal also measures some reversibly bound methylglyoxal.

  9. The use of human amniotic fluid mesenchymal stem cells as the feeder layer to establish human embryonic stem cell lines.

    PubMed

    Soong, Yung-Kwei; Huang, Shang-Yu; Yeh, Chiu-Hsiang; Wang, Tzu-Hao; Chang, Kuo-Hsuan; Cheng, Po-Jen; Shaw, S W Steven

    2015-12-01

    Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into the three germ layers and possibly all tissues of the human body. To fulfil the clinical potentials for cell-based therapy, banks of hESC lines that express different combinations of the major histocompatibility genes should be established, preferably without exposing such cells to animal cells and proteins. In this study, we tested human amniotic fluid mesenchymal stem cells (AFMSCs) as feeder cells to support the growth of hESCs. Our results indicated that mitomycin-treated AFMSCs were able to support the newly established hESC lines CGLK-1 and CGLK-2. The hESC colonies cultured on AFMSCs expressed alkaline phosphatase (ALK-P), SSEA-4, TRA-1-60, TRA-1-81, Oct-4, Nanog and Sox-2, which are markers for undifferentiated hESCs. Chromosomal analyses of both hESC lines, CGLK-1 and CGLK-2, which were cultured on AFMSC feeders for 22 and 14 passages, respectively, were confirmed to be normal karyotypes (46, XX). The ability of AFMSCs as feeder cells to maintain the undifferentiated growth and pluripotency of hESCs was confirmed by in vivo formation of teratomas derived on AFMSC hESCs in severe combined immune-compromised mice. The use of AFMSCs for feeder cells to culture hESCs has several advantages, in that AFMSCs are not tumourigenic and can be expanded extensively with a short doubling time.

  10. Human norovirus infection of caco-2 cells grown as a three-dimensional tissue structure.

    PubMed

    Straub, Timothy M; Bartholomew, Rachel A; Valdez, Catherine O; Valentine, Nancy B; Dohnalkova, Alice; Ozanich, Richard M; Bruckner-Lea, Cynthia J; Call, Douglas R

    2011-06-01

    Human norovirus (hNoV) infectivity was studied using a three-dimensional model of large intestinal epithelium. Large intestine Caco-2 cells were grown in rotating wall vessel bioreactors for 18-21 days at 37 degrees C and then transferred to 24-well tissue culture plates where they were infected with GI.1 and GII.4 human noroviruses collected from human challenge trials and various outbreak settings, respectively. Compared with uninfected cells, transmission micrographs of norovirus-infected cells displayed evidence of shortening or total loss of apical microvilli, and vacuolization. Quantitative reverse transcription real-time PCR (qRT-PCR) indicated an approximate 2-3 log10 increase in viral RNA copies for the infected cells. A passage experiment examined both the ability for continued viral RNA and viral antigen detection. In the passaged samples 1.01x10(6) copies ml(-1) were detected by qRT-PCR. Immune electron microscopy using primary antibody to hNoV GI.1 capsids in conjunction with 6 nm gold-labelled secondary antibodies was performed on crude cellular lysates. Localization of antibody was observed in infected but not for uninfected cells. Our present findings, coupled with earlier work with the three-dimensional small intestinal INT407 model, demonstrate the utility of 3-D cell culture methods to develop infectivity assays for enteric viruses that do not readily infect mammalian cell cultures. PMID:21942189

  11. Mesenchymal traits are selected along with stem features in breast cancer cells grown as mammospheres

    PubMed Central

    Borgna, Silvia; Armellin, Michela; di Gennaro, Alessandra; Maestro, Roberta; Santarosa, Manuela

    2012-01-01

    Increasing evidence indicates that invasive properties of breast cancers rely on gain of mesenchymal and stem features, which has suggested that the dual targeting of these phenotypes may represent an appealing therapeutic strategy. It is known that the fraction of stem cells can be enriched by culturing breast cancer cells as mammospheres (MS), but whether these pro-stem conditions favor also the expansion of cells provided of mesenchymal features is still undefined. In the attempt to shed light on this issue, we compared the phenotypes of a panel of 10 breast cancer cell lines representative of distinct subtypes (luminal, HER2-positive, basal-like and claudin-low), grown in adherent conditions and as mammospheres. Under MS-proficient conditions, the increment in the fraction of stem-like cells was associated to upregulation of the mesenchymal marker Vimentin and downregulation of the epithelial markers expressed by luminal cells (E-cadherin, KRT18, KRT19, ESR1). Luminal cells tended also to upregulate the myoepithelial marker CD10. Taken together, our data indicate that MS-proficient conditions do favor mesenchymal/myoepithelial features, and indicate that the use of mammospheres as an in vitro tumor model may efficiently allow the exploitation of therapeutic approaches aimed at targeting aggressive tumors that have undergone epithelial-to-mesenchymal transition. PMID:23095640

  12. Human norovirus infection of caco-2 cells grown as a three-dimensional tissue structure.

    PubMed

    Straub, Timothy M; Bartholomew, Rachel A; Valdez, Catherine O; Valentine, Nancy B; Dohnalkova, Alice; Ozanich, Richard M; Bruckner-Lea, Cynthia J; Call, Douglas R

    2011-06-01

    Human norovirus (hNoV) infectivity was studied using a three-dimensional model of large intestinal epithelium. Large intestine Caco-2 cells were grown in rotating wall vessel bioreactors for 18-21 days at 37 degrees C and then transferred to 24-well tissue culture plates where they were infected with GI.1 and GII.4 human noroviruses collected from human challenge trials and various outbreak settings, respectively. Compared with uninfected cells, transmission micrographs of norovirus-infected cells displayed evidence of shortening or total loss of apical microvilli, and vacuolization. Quantitative reverse transcription real-time PCR (qRT-PCR) indicated an approximate 2-3 log10 increase in viral RNA copies for the infected cells. A passage experiment examined both the ability for continued viral RNA and viral antigen detection. In the passaged samples 1.01x10(6) copies ml(-1) were detected by qRT-PCR. Immune electron microscopy using primary antibody to hNoV GI.1 capsids in conjunction with 6 nm gold-labelled secondary antibodies was performed on crude cellular lysates. Localization of antibody was observed in infected but not for uninfected cells. Our present findings, coupled with earlier work with the three-dimensional small intestinal INT407 model, demonstrate the utility of 3-D cell culture methods to develop infectivity assays for enteric viruses that do not readily infect mammalian cell cultures.

  13. Enhanced Migration of Neural Stem Cells by Microglia Grown on a Three-Dimensional Graphene Scaffold.

    PubMed

    Jiang, Ziyun; Song, Qin; Tang, Mingliang; Yang, Lingyan; Cheng, Yilin; Zhang, Min; Xu, Dongsheng; Cheng, Guosheng

    2016-09-28

    One of the key challenges in engineering neural tissues for cell-based therapies is to develop a biocompatible scaffold material to direct neural stem cell (NSC) behaviors. One great advantage for a scaffold would be to induce NSC migration toward pathological sites during regeneration and repair. In particular, the inflammatory responses in the pathological zone, which are mainly mediated by microglia in the central nervous system, affect the repair capacity of NSCs through NSC migration. Recently, graphene was used as a neural interface and scaffold material, but few studies have addressed the relationship between microglia and NSCs in a graphene culture system. In this study, we used a combination of immunofluorescence, Western blotting, enzyme-linked immunosorbent assays, and scanning electron microscopy to investigate how conditioned medium (CM) produced from microglia grown on two-dimensional graphene (2D-G) films or three-dimensional graphene (3D-G) foams govern NSC migration. The results revealed that the CM produced by microglia grown in 3D-G cultures could promote neurosphere formation, facilitate NSC migration from the neurospheres, and increase single cell polarization by activating the stromal cell-derived factor 1 α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) signaling pathway and enhancing cell adhesion on the substrate. By contrast, the 2D-G CM failed to achieve these results. Our study suggests the great potential of 3D-G as a neural scaffold for NSC-based therapy in tissue engineering and regenerative medicine. PMID:27589088

  14. Characterization of Epitaxial Film Silicon Solar Cells Grown on Seeded Display Glass: Preprint

    SciTech Connect

    Young, D. L.; Grover, S.; Teplin, C.; Stradins, P.; LaSalvia, V.; Chuang, T. K.; Couillard, J. G.; Branz, H. M.

    2012-06-01

    We report characterizations of epitaxial film crystal silicon (c-Si) solar cells with open-circuit voltages (Voc) above 560 mV. The 2-um absorber cells are grown by low-temperature (<750 degrees C) hot-wire CVD (HWCVD) on Corning EAGLE XG display glass coated with a layer-transferred (LT) Si seed. The high Voc is a result of low-defect epitaxial Si (epi-Si) growth and effective hydrogen passivation of defects. The quality of HWCVD epitaxial growth on seeded glass substrates depends on the crystallographic quality of the seed and the morphology of the epitaxial growth surface. Heterojunction devices consist of glass/c-Si LT seed/ epi n+ Si:P/epi n- Si:P/intrinsic a-Si:H/p+ a-Si:H/ITO. Similar devices grown on electronically 'dead' n+ wafers have given Voc {approx}630 mV and {approx}8% efficiency with no light trapping features. Here we study the effects of the seed surface polish on epi-Si quality, how hydrogenation influences the device character, and the dominant junction transport physics.

  15. Comparison of Chloroflexus aurantiacus strain J-10-fl proteomes of cells grown chemoheterotrophically and photoheterotrophically

    SciTech Connect

    Cao, Li; Bryant, Donald A.; Schepmoes, Athena A.; Vogl, Kajetan; Smith, Richard D.; Lipton, Mary S.; Callister, Stephen J.

    2012-01-17

    Chloroflexus aurantiacus J-10-fl is a thermophilic green bacterium, a filamentous anoxygenic phototroph, and the model organism of the phylum Chloroflexi. We applied high-throughput, liquid chromatography-mass spectrometry in a global quantitative proteomics investigation of C. aurantiacus cells grown under oxic (chemoorganoheterotrophically) and anoxic (photoorganoheterotrophically) redox states. Our global analysis identified 13,524 high-confidence peptides that matched to 1,286 annotated proteins, 242 of which were either uniquely identified or significantly increased in abundance under anoxic culture conditions. Fifty-three of the 242 proteins are previously characterized photosynthesis-related proteins, including chlorosome proteins, proteins involved in the bacteriochlorophyll biosynthesis, 3-hydroxypropionate (3-OHP) CO2 fixation pathway, and components of electron transport chains. The remaining 190 proteins have not previously been reported. Of these, five proteins were found to be encoded by genes from a novel operon and observed only in photoheterotrophically grown cells. These proteins candidates may prove useful in further deciphering the phototrophic physiology of C. aurantiacus and other filamentous anoxygenic phototrophs.

  16. Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds.

    PubMed

    Yeh, Hsi-Yi; Lin, Ting-Yu; Lin, Chen-Huan; Yen, B Linju; Tsai, Ching-Lin; Hsu, Shan-Hui

    2013-01-01

    Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-β3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

  17. Effects of low-level laser therapy on human osteoblastic cells grown on titanium.

    PubMed

    Petri, Alice Dias; Teixeira, Lucas Novaes; Crippa, Grasiele Edilaine; Beloti, Marcio Mateus; de Oliveira, Paulo Tambasco; Rosa, Adalberto Luiz

    2010-01-01

    The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation. PMID:21271038

  18. Stimulation of Cell Elongation by Tetraploidy in Hypocotyls of Dark-Grown Arabidopsis Seedlings

    PubMed Central

    Narukawa, Hideki; Yokoyama, Ryusuke; Komaki, Shinichiro; Sugimoto, Keiko; Nishitani, Kazuhiko

    2015-01-01

    Plant size is largely determined by the size of individual cells. A number of studies showed a link between ploidy and cell size in land plants, but this link remains controversial. In this study, post-germination growth, which occurs entirely by cell elongation, was examined in diploid and autotetraploid hypocotyls of Arabidopsis thaliana (L.) Heynh. Final hypocotyl length was longer in tetraploid plants than in diploid plants, particularly when seedlings were grown in the dark. The longer hypocotyl in the tetraploid seedlings developed as a result of enhanced cell elongation rather than by an increase in cell number. DNA microarray analysis showed that genes involved in the transport of cuticle precursors were downregulated in a defined region of the tetraploid hypocotyl when compared to the diploid hypocotyl. Cuticle permeability, as assessed by toluidine-blue staining, and cuticular structure, as visualized by electron microscopy, were altered in tetraploid plants. Taken together, these data indicate that promotion of cell elongation is responsible for ploidy-dependent size determination in the Arabidopsis hypocotyl, and that this process is directly or indirectly related to cuticular function. PMID:26244498

  19. Electron-cytochemical study of Ca2+ in cotyledon cells of soybean seedlings grown in microgravity

    NASA Technical Reports Server (NTRS)

    Nedukha, O.; Brown, C. S.; Kordyum, E.; Piastuch, W. C.; Guikema, J. A. (Principal Investigator)

    1999-01-01

    Microgravity and horizontal clinorotation are known to cause the rearrangement of the structural-functional organization of plant cells, leading to accelerated aging. Altered gravity conditions resulted in an increase in the droplets volume in cells and the destruction of chloroplast structure in Arabidopsis thaliana plants, an enhancement of cytosolic autophagaous processes, an increase in the respiration rate and a greater number of multimolecular forms of succinate- and malate dehydrogenases in cells of the Funaria hygrometrica protonema and Chlorella vulgaris, and changes in calcium balance of cells. Because ethylene is known to be involved in cell aging and microgravity appears to speed the process, and because soybean seedlings grown in space produce higher ethylene levels we asked: 1) does an acceleration of soybean cotyledon cell development and aging occur in microgravity? 2) what roles do Ca2+ ions and the enhanced ethylene level play in these events? Therefore, the goal of our investigation was to examine of the interaction of microgravity and ethylene on the localization of Ca2+ in cotyledon mesophyll of soybean seedlings.

  20. Differential Response in Downstream Processing of CHO Cells Grown Under Mild Hypothermic Conditions

    PubMed Central

    Tait, Andrew S; Tarrant, Richard D R; Velez-Suberbie, M Lourdes; Spencer, Daniel I R; Bracewell, Daniel G

    2013-01-01

    The manufacture of complex therapeutic proteins using mammalian cells is well established, with several strategies developed to improve productivity. The application of sustained mild hypothermic conditions during culture has been associated with increases in product titer and improved product quality. However, despite associated cell physiological effects, very few studies have investigated the impact on downstream processing (DSP). Characterization of cells grown under mild hypothermic conditions demonstrated that the stationary phase was prolonged by delaying the onset of apoptosis. This enabled cells to maintain viability for extended periods and increase volumetric productivity from 0.74 to 1.02 g L−1. However, host cell proteins, measured by ELISA, increased by ∼50%, attributed to the extended time course and higher peak and harvest cell densities. The individual components making up this impurity, as determined by SELDI-TOF MS and 2D-PAGE, were shown to be largely comparable. Under mild hypothermic conditions, cells were less shear sensitive than those maintained at 37°C, enhancing the preliminary primary recovery step. Adaptive changes in membrane fluidity were further investigated by adopting a pronounced temperature shift immediately prior to primary recovery and the improvement observed suggests that such a strategy may be implementable when shear sensitivity is of concern. Early and late apoptotic cells were particularly susceptible to shear, at either temperature, even under the lowest shear rate investigated. These findings demonstrate the importance of considering the impact of cell culture strategies and cell physiology on DSP, by implementing a range of experimental methods for process characterization. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:688–696, 2013 PMID:23636936

  1. Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays

    PubMed Central

    Jain, Tilak; McBride, Ryan; Head, Steven; Saez, Enrique

    2010-01-01

    High-throughput cell-based screens of genome-size collections of cDNAs and siRNAs have become a powerful tool to annotate the mammalian genome, enabling the discovery of novel genes associated with normal cellular processes and pathogenic states, and the unraveling of genetic networks and signaling pathways in a systems biology approach. However, the capital expenses and the cost of reagents necessary to perform such large screens have limited application of this technology. Efforts to miniaturize the screening process have centered on the development of cellular microarrays created on microscope slides that use chemical means to introduce exogenous genetic material into mammalian cells. While this work has demonstrated the feasibility of screening in very small formats, the use of chemical transfection reagents (effective only in a subset of cell lines and not on primary cells) and the lack of defined borders between cells grown in adjacent microspots containing different genetic material (to prevent cell migration and to aid spot location recognition during imaging and phenotype deconvolution) have hampered the spread of this screening technology. Here, we describe proof-of-principles experiments to circumvent these drawbacks. We have created microwell arrays on an electroporation-ready transparent substrate and established procedures to achieve highly efficient parallel introduction of exogenous molecules into human cell lines and primary mouse macrophages. The microwells confine cells and offer multiple advantages during imaging and phenotype analysis. We have also developed a simple method to load this 484-microwell array with libraries of nucleic acids using a standard microarrayer. These advances can be elaborated upon to form the basis of a miniaturized high-throughput functional genomics screening platform to carry out genome-size screens in a variety of mammalian cells that may eventually become a mainstream tool for life science research. PMID:20024036

  2. Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays.

    PubMed

    Jain, Tilak; McBride, Ryan; Head, Steven; Saez, Enrique

    2009-12-21

    High-throughput cell-based screens of genome-size collections of cDNAs and siRNAs have become a powerful tool to annotate the mammalian genome, enabling the discovery of novel genes associated with normal cellular processes and pathogenic states, and the unravelling of genetic networks and signaling pathways in a systems biology approach. However, the capital expenses and the cost of reagents necessary to perform such large screens have limited application of this technology. Efforts to miniaturize the screening process have centered on the development of cellular microarrays created on microscope slides that use chemical means to introduce exogenous genetic material into mammalian cells. While this work has demonstrated the feasibility of screening in very small formats, the use of chemical transfection reagents (effective only in a subset of cell lines and not on primary cells) and the lack of defined borders between cells grown in adjacent microspots containing different genetic material (to prevent cell migration and to aid spot location recognition during imaging and phenotype deconvolution) have hampered the spread of this screening technology. Here, we describe proof-of-principles experiments to circumvent these drawbacks. We have created microwell arrays on an electroporation-ready transparent substrate and established procedures to achieve highly efficient parallel introduction of exogenous molecules into human cell lines and primary mouse macrophages. The microwells confine cells and offer multiple advantages during imaging and phenotype analysis. We have also developed a simple method to load this 484-microwell array with libraries of nucleic acids using a standard microarrayer. These advances can be elaborated upon to form the basis of a miniaturized high-throughput functional genomics screening platform to carry out genome-size screens in a variety of mammalian cells that may eventually become a mainstream tool for life science research.

  3. Label-free optical detection of cells grown in 3D silicon microstructures.

    PubMed

    Merlo, Sabina; Carpignano, Francesca; Silva, Gloria; Aredia, Francesca; Scovassi, A Ivana; Mazzini, Giuliano; Surdo, Salvatore; Barillaro, Giuseppe

    2013-08-21

    We demonstrate high aspect-ratio photonic crystals that could serve as three-dimensional (3D) microincubators for cell culture and also provide label-free optical detection of the cells. The investigated microstructures, fabricated by electrochemical micromachining of standard silicon wafers, consist of periodic arrays of silicon walls separated by narrow deeply etched air-gaps (50 μm high and 5 μm wide) and feature the typical spectral properties of photonic crystals in the wavelength range 1.0-1.7 μm: their spectral reflectivity is characterized by wavelength regions where reflectivity is high (photonic bandgaps), separated by narrow wavelength regions where reflectivity is very low. In this work, we show that the presence of cells, grown inside the gaps, strongly affects light propagation across the photonic crystal and, therefore, its spectral reflectivity. Exploiting a label-free optical detection method, based on a fiberoptic setup, we are able to probe the extension of cells adherent to the vertical silicon walls with a non-invasive direct testing. In particular, the intensity ratio at two wavelengths is the experimental parameter that can be well correlated to the cell spreading on the silicon wall inside the gaps.

  4. Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space.

    PubMed

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki; Kotake, Toshihisa; Yamazaki, Takashi; Higashibata, Akira; Ishioka, Noriaki; Shimazu, Toru; Fukui, Keiji; Osada, Ikuko; Kasahara, Haruo; Kamada, Motoshi

    2015-01-01

    Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.

  5. Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space

    PubMed Central

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki; Kotake, Toshihisa; Yamazaki, Takashi; Higashibata, Akira; Ishioka, Noriaki; Shimazu, Toru; Fukui, Keiji; Osada, Ikuko; Kasahara, Haruo; Kamada, Motoshi

    2015-01-01

    Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions. PMID:26378793

  6. Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space.

    PubMed

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki; Kotake, Toshihisa; Yamazaki, Takashi; Higashibata, Akira; Ishioka, Noriaki; Shimazu, Toru; Fukui, Keiji; Osada, Ikuko; Kasahara, Haruo; Kamada, Motoshi

    2015-01-01

    Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions. PMID:26378793

  7. Xylem Development and Cell Wall Changes of Soybean Seedlings Grown in Space

    PubMed Central

    de Micco, Veronica; Aronne, Giovanna; Joseleau, Jean-Paul; Ruel, Katia

    2008-01-01

    Background and Aims Plants growing in altered gravity conditions encounter changes in vascular development and cell wall deposition. The aim of this study was to investigate xylem anatomy and arrangement of cellulose microfibrils in vessel walls of different organs of soybean seedlings grown in Space. Methods Seeds germinated and seedlings grew for 5 d in Space during the Foton-M2 mission. The environmental conditions, other than gravity, of the ground control repeated those experienced in orbit. The seedlings developed in space were compared with those of the control test on the basis of numerous anatomical and ultrastructural parameters such as number of veins, size and shape of vessel lumens, thickness of cell walls and deposition of cellulose microfibrils. Key Results Observations made with light, fluorescence and transmission electron microscopy, together with the quantification of the structural features through digital image analysis, showed that the alterations due to microgravity do not occur at the same level in the various organs of soybean seedlings. The modifications induced by microgravity or by the indirect effect of space-flight conditions, became conspicuous only in developing vessels at the ultrastructural level. The results suggested that the orientation of microfibrils and their assembly in developing vessels are perturbed by microgravity at the beginning of wall deposition, while they are still able to orient and arrange in thicker and ordered structures at later stages of secondary wall deposition. Conclusions The process of proper cell-wall building, although not prevented, is perturbed in Space at the early stage of development. This would explain the almost unaltered anatomy of mature structures, accompanied by a slower growth observed in seedlings grown in Space than on Earth. PMID:18252765

  8. Spheroid formation and enhanced cardiomyogenic potential of adipose-derived stem cells grown on chitosan.

    PubMed

    Liu, Bing-Hsien; Yeh, Hsi-Yi; Lin, Yu-Chun; Wang, Min-Hsiung; Chen, David C; Lee, Bo-Hua; Hsu, Shan-Hui

    2013-02-01

    Mesenchymal stem cells may differentiate into cardiomyocytes and participate in local tissue repair after heart injury. In the current study, rat adipose-derived adult stem cells (ASCs) grown on chitosan membranes were observed to form cell spheroids after 3 days. The cell seeding density and surface modification of chitosan with Arg-Gly-Asp-containing peptide had an influence on the sizes of ASC spheroids. In the absence of induction, these spheroids showed an increased level of cardiac marker gene expression (Gata4, Nkx2-5, Myh6, and Tnnt2) more than 20-fold versus cells on the tissue culture polystyrene (TCPS) dish. Induction by 5-azacytidine or p38 MAP kinase inhibitor (SB202190) did not further increase the cardiac marker gene expression of these spheroids. Moreover, the enhanced cardiomyogenic potential of the spheroids was highly associated with the chitosan substrates. When ASC spheroids were plated onto TCPS with either basal or cardiac induction medium for 9 days, the spheroids spread into a monolayer and the positive effect on cardiomyogenic marker gene expression disappeared. The possible role of calcium ion and the up-regulation of adhesion molecule P-selectin and chemokine receptor Cxcr4 were demonstrated in ASC spheroids. Applying these spheroids to the chronic myocardial infarction animal model showed better functional recovery versus single cells after 12 weeks. Taken together, this study suggested that the ASC spheroids on chitosan may form as a result of calcium ion signaling, and the transplantation of these spheroids may offer a simple method to enhance the efficiency of stem cell-based therapy in myocardial infarction. PMID:23514754

  9. Electrochemically grown ZnO nanorods for hybrid solar cell applications

    SciTech Connect

    Hames, Yakup; Alpaslan, Zuehal; Koesemen, Arif; San, Sait Eren; Yerli, Yusuf

    2010-03-15

    A hybrid solar cell is designed and proposed as a feasible and reasonable alternative, according to acquired efficiency with the employment of zinc oxide (ZnO) nanorods and ZnO thin films at the same time. Both of these ZnO structures were grown electrochemically and poly(3-hexylthiophene):phenyl-C61-butyric acid methyl ester; (P3HT:PCBM) was used as an active polymer blend, which was found to be compatible to prepared indium-tin-oxide (ITO) substrate base. This ITO base was introduced with mentioned ZnO structure in such a way that, the most efficient configuration was optimized to be ITO/ZnO film/ZnO nanorod/P3HT: PCBM/Ag. Efficiency of this optimized device is found to be 2.44%. All ZnO works were carried out electrochemically, that is indeed for the first time and at relatively lower temperatures. (author)

  10. Sensitivity of urine-grown cells of Providencia stuartii to antiseptics.

    PubMed

    Thomas, B; Sykes, L; Stickler, D J

    1978-10-01

    Urine-grown cultures of 23 clinical isolates of Gram-negative bacteria having a range of minimum inhibitory concentration values for chorhexidine were challenged with various concentrations of this antiseptic. The results suggest that cells of Providencia stuartii, in particular, exhibit a considerable degree of resistance to chlorhexidine under these conditions, concentrations of up to 10 000--20 000 microgram/ml of urine being necessary to produce complete loss of viability of such cultures. Of the other two antiseptics tested, phenoxyethanol proved to be the more effective, the recommended use concentration of 2% v/v producing reductions in viable counts of greater than six logarithms in all the strains examined. It is suggested that phenoxyethanol may be a suitable alternative to the cationic agents for use in antiseptic policies for bladder management of urinary tract infections with Providencia stuartii.

  11. Systematic process development towards high performance transferred thin silicon solar cells based on epitaxially grown absorbers

    NASA Astrophysics Data System (ADS)

    Murcia Salazar, Clara Paola

    ). First principles modeling, however, predicts that efficiencies of 20+% are achievable with less than 20 mum of c-Si. In addition to a high voltage design, this work reports state of the art epitaxial c-Si solar cell performance and a path towards 20+%-efficient transferred epitaxial solar cells. The design and fabrication approach is based on high open circuit voltage first, high short circuit current second. A first design is a thin solar cell grown on a conductive silicon wafer. This structure allows developing processes to increase bulk lifetime and reduce surface recombination. Important processes that can be used for a transferred solar cell such as increased fill factor (FF) are developed at this stage. A second design is based on the use of a separation layer prior to the solar cell growth. We achieve a comparable performance with the second design. A third design includes the transfer of the solar cell to a secondary substrate. Initial processing development is reported for the transferred solar cells. Improvements in solar cell critical parameters have been characterized with a combination of predictive modeling and solar cell diagnostic tools such as quantum efficiency and voltage measurements. Fabrication processes have been developed to improve solar cell performance. The combination of process development, test structures, systematic fabrication, testing and analysis concludes with a path to high voltage, transferred thin c-Si solar cells towards 20+% efficiencies.

  12. Abnormal physiological properties and altered cell wall composition in Streptococcus pneumoniae grown in the presence of clavulanic acid.

    PubMed Central

    Severin, A; Severina, E; Tomasz, A

    1997-01-01

    Subinhibitory concentrations of clavulanate caused premature induction of stationary-phase autolysis, sensitization to lysozyme, and reductions in the MICs of deoxycholate and penicillin for Streptococcus pneumoniae. In the range of clavulanate concentrations producing these effects, this beta-lactam compound was selectively bound to PBP 3. Cell walls isolated from pneumococci grown in the presence of clavulanate showed increased sensitivity to the hydrolytic action of purified pneumococcal autolysin in vitro. High-performance liquid chromatography analysis of the peptidoglycan isolated from the clavulanate-grown cells showed major qualitative and quantitative changes in stem peptide composition, the most striking feature of which was the accumulation of peptide species carrying intact D-alanyl-D-alanine residues at the carboxy termini. The altered biological and biochemical properties of the clavulanate-grown pneumococci appear to be the consequences of suppressed D,D-carboxypeptidase activity. PMID:9055983

  13. Berry extracts exert different antiproliferative effects against cervical and colon cancer cells grown in vitro.

    PubMed

    McDougall, Gordon J; Ross, Heather A; Ikeji, Magnus; Stewart, Derek

    2008-05-14

    Polyphenol-rich berry extracts were screened for their antiproliferative effectiveness using human cervical cancer (HeLa) cells grown in microtiter plates. Rowan berry, raspberry, lingonberry, cloudberry, arctic bramble, and strawberry extracts were effective but blueberry, sea buckthorn, and pomegranate extracts were considerably less effective. The most effective extracts (strawberry > arctic bramble > cloudberry > lingonberry) gave EC 50 values in the range of 25-40 microg/(mL of phenols). These extracts were also effective against human colon cancer (CaCo-2) cells, which were generally more sensitive at low concentrations but conversely less sensitive at higher concentrations. The strawberry, cloudberry, arctic bramble, and the raspberry extracts share common polyphenol constituents, especially the ellagitannins, which have been shown to be effective antiproliferative agents. However, the components underlying the effectiveness of the lingonberry extracts are not known. The lingonberry extracts were fractionated into anthocyanin-rich and tannin-rich fractions by chromatography on Sephadex LH-20. The anthocyanin-rich fraction was considerably less effective than the original extract, whereas the antiproliferative activity was retained in the tannin-rich fraction. The polyphenolic composition of the lingonberry extract was assessed by liquid chromatography-mass spectrometry and was similar to previous reports. The tannin-rich fraction was almost entirely composed of procyanidins of linkage type A and B. Therefore, the antiproliferative activity of lingonberry was caused predominantly by procyanidins. PMID:18412361

  14. Radial junction amorphous silicon solar cells on PECVD-grown silicon nanowires.

    PubMed

    Yu, Linwei; O'Donnell, Benedict; Foldyna, Martin; Roca i Cabarrocas, Pere

    2012-05-17

    Constructing radial junction hydrogenated amorphous silicon (a-Si:H) solar cells on top of silicon nanowires (SiNWs) represents a promising approach towards high performance and cost-effective thin film photovoltaics. We here develop an all-in situ strategy to grow SiNWs, via a vapour-liquid-solid (VLS) mechanism on top of ZnO-coated glass substrate, in a plasma-enhanced chemical vapour deposition (PECVD) reactor. Controlling the distribution of indium catalyst drops allows us to tailor the as-grown SiNW arrays into suitable size and density, which in turn results in both a sufficient light trapping effect and a suitable arrangement allowing for conformal coverage of SiNWs by subsequent a-Si:H layers. We then demonstrate the fabrication of radial junction solar cells and carry on a parametric study designed to shed light on the absorption and quantum efficiency response, as functions of the intrinsic a-Si:H layer thickness and the density of SiNWs. These results lay a solid foundation for future structural optimization and performance ramp-up of the radial junction thin film a-Si:H photovoltaics.

  15. Tilted bulk heterojunction organic photovoltaic cells grown by oblique angle deposition

    NASA Astrophysics Data System (ADS)

    Li, Ning; Forrest, Stephen R.

    2009-09-01

    We demonstrate small molecule bulk heterojunction organic photovoltaic cells using oblique angle vacuum deposition. Obliquely deposited donor chloroaluminum phthalocyanine (ClAlPc) films on indium tin oxide have surface feature sizes of ˜30 nm, resulting in ClAlPc/C60 donor-acceptor heterojunctions (HJs) with approximately twice the interface area of HJs grown at normal incidence. This results in nearly twice the external quantum efficiency in the ClAlPc absorption band compared with analogous, planar HJs. The efficiency increase is attributed to the increased surface area presented by the donor-acceptor junction to the incident illumination by ClAlPc protrusions lying obliquely to the substrate plane formed during deposition. The power conversion efficiency improves from (2.0±0.1)% to (2.8±0.1)% under 1 sun, AM 1.5G simulated solar illumination. Similarly, the power efficiency of copper phthalocyanine/C60 organic photovoltaic cells is increased from (1.3±0.1)% to (1.7±0.1)%.

  16. GaSb thermophotovoltaic cells grown on GaAs by molecular beam epitaxy using interfacial misfit arrays

    SciTech Connect

    Juang, Bor-Chau Laghumavarapu, Ramesh B.; Foggo, Brandon J.; Lin, Andrew; Simmonds, Paul J.; Liang, Baolai; Huffaker, Diana L.

    2015-03-16

    There exists a long-term need for foreign substrates on which to grow GaSb-based optoelectronic devices. We address this need by using interfacial misfit arrays to grow GaSb-based thermophotovoltaic cells directly on GaAs (001) substrates and demonstrate promising performance. We compare these cells to control devices grown on GaSb substrates to assess device properties and material quality. The room temperature dark current densities show similar characteristics for both cells on GaAs and on GaSb. Under solar simulation the cells on GaAs exhibit an open-circuit voltage of 0.121 V and a short-circuit current density of 15.5 mA/cm{sup 2}. In addition, the cells on GaAs substrates maintain 10% difference in spectral response to those of the control cells over a large range of wavelengths. While the cells on GaSb substrates in general offer better performance than the cells on GaAs substrates, the cost-savings and scalability offered by GaAs substrates could potentially outweigh the reduction in performance. By further optimizing GaSb buffer growth on GaAs substrates, Sb-based compound semiconductors grown on GaAs substrates with similar performance to devices grown directly on GaSb substrates could be realized.

  17. Glucose-triggered signalling in Saccharomyces cerevisiae: different requirements for sugar phosphorylation between cells grown on glucose and those grown on non-fermentable carbon sources.

    PubMed

    Pernambuco, M B; Winderickx, J; Crauwels, M; Griffioen, G; Mager, W H; Thevelein, J M

    1996-07-01

    Addition of glucose or fructose to cells of the yeast Saccharomyces cerevisiae grown on a nonfermentable carbon source triggers within a few minutes posttranslational activation of trehalase, repression of the CTT1 (catalase) and SSA3 (Hsp70) genes, and induction of the ribosomal protein genes RPL1, RPL25 and RPS33. By using appropriate sugar kinase mutants, it was shown that rapid glucose- or fructose-induced activation of trehalase requires phosphorylation of the sugar. On the other hand, partial induction of RPL1, RPL25 and RPS33 as well as partial repression of CTT1 and SSA3 were observed in the absence of sugar phosphorylation. In glucose-grown nitrogen-starved yeast cells readdition of a nitrogen source triggers activation of trehalase in a glucose- or fructose-dependent way, but with no apparent requirements for phosphorylation of the sugar. Repression of CTT1 and SSA3 under the same conditions was also largely dependent on the presence of the sugar and also in these cases there was a strong effect when the sugar could not be phosphorylated. Nitrogen induction of RPL1, RPL25 and RPS33 was much less dependent on the presence of the sugar, and only phosphorylated sugar caused a further increase in expression. These results show that two glucose-dependent signalling pathways, which can be distinguished on the basis of their requirement for glucose phosphorylation, appear to be involved in activation of trehalase, repression of CTT1 and SSA3 and induction of ribosomal protein genes. They also show that nutrient-induced repression of CTT1 and SSA3 is not a response to improvement of the growth conditions because the addition of nonmetabolizable sugar does not ameliorate the growth conditions. Similarly, the upshift in ribosomal protein synthesis cannot be a response to increased availability of energy or biosynthetic capacity derived from glucose, but it is apparently triggered to a significant extent by specific detection of glucose as such.

  18. Information on Stem Cell Research

    MedlinePlus

    ... Enhancing Diversity Find People About NINDS Information on Stem Cell Research Research @ NINDS Stem Cell Highlights Submit a hESC ... found here: Human Induced Pluripotent Stem Cells NINDS Stem Cell Research on Campus The Intramural Research Program of NINDS ...

  19. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    PubMed

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-01-01

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. PMID:26840224

  20. Global gene expression profile of osteoblast-like cells grown on polyester copolymer scaffolds.

    PubMed

    Idris, Shaza B; Bolstad, Anne Isine; Ibrahim, Salah O; Dånmark, Staffan; Finne-Wistrand, Anna; Albertsson, Ann-Christine; Arvidson, Kristina; Mustafa, Kamal

    2011-11-01

    One of the principal goals in tissue engineering is to produce scaffold materials that will guide cells to differentiate and regenerate functional replacement tissue at the site of injury. Poly(l-lactide-co-1,5-dioxepan-2-one) [Poly(LLA-co-DXO)], a potential scaffolding material for bone tissue engineering, has high hydrophilicity. Previous in vitro studies using human osteoblast-like cells (HOBs) demonstrated greater cytocompatibility and enhanced osteogenic differentiation when HOBs were seeded onto Poly(LLA-co-DXO) compared to Poly(l-lactide) [P(LLA)] scaffolds. The aim of the study was to identify the gene expression profiles of HOBs obtained from alveolar bone and grown on Poly(LLA-co-DXO) biodegradable polymer scaffolds compared to P(LLA) one. Illumina HumanWG-6 v3.0 Expression BeadChips were used for the gene expression analysis. Several genes were found as differentially expressed at 24 h and at 21 days. Expression of genes related to cell adhesion, cytoskeleton, antiapoptosis, proliferation, and bone mineralization was influenced by adding the monomer 1,5-dioxepan-2-one to the L-lactide. Genes related to three biological pathways involving Integrin, Notch, and Ras were found to be upregulated. For selected genes, results were confirmed by quantitative reverse transcriptase-polymerase chain reaction. Further, calcium content analysis revealed a significant enhancement of calcium deposition on both tested scaffolds. This observation was confirmed by Von Kossa and Alizarin Red S staining. Findings of this study are relevant to a better understanding of the molecular mechanisms underlying the behavior of HOBs in bone regenerative procedure.

  1. A simple pneumatic device for plunge-freezing cells grown on electron microscopy grids.

    PubMed

    Cole, R; Matuszek, G; See, C; Rieder, C L

    1990-10-01

    A detailed design for a simple and inexpensive variable-speed (1.0-5.8 m s-1) pneumatic plunge-freezing device is presented. Cultured cells, grown on Formvar-coated 75-mesh gold finder grids, are pneumatically driven into a stirring mixture of propane/isopentane (3:1) cooled by liquid nitrogen (LN2). Premature freezing of the sample in the cryogenic vapors above the cryogen is prevented by plunging through an entry tube into an insulating box, to which a partial vacuum is applied. The cryogenic vapors are drafted into the box at the level of the liquid cryogen by the vacuum, thereby preventing a layer of cold gas from collecting above the cryogen. To prevent the sample from thawing during transfer from the cryogen to the substitution medium, the box top is removed and compressed air is forced through a corrugated tube running the length of the box. The resulting boiling LN2 creates an atmosphere below -120 degrees C in which the transfer can be accomplished. PMID:2213239

  2. Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells

    SciTech Connect

    Miyazaki, Takamichi; Futaki, Sugiko; Hasegawa, Kouichi; Kawasaki, Miwa; Sanzen, Noriko; Hayashi, Maria; Kawase, Eihachiro; Sekiguchi, Kiyotoshi Nakatsuji, Norio; Suemori, Hirofumi

    2008-10-10

    Human embryonic stem cells (hESCs) are thought to be a promising cell source for cell transplantation therapy. For such a clinical application, the hESCs should be manipulated using appropriate and qualified materials. In this study, we examined the efficacy of recombinant human laminin (rhLM) isoforms on the undifferentiated growth of hESCs. We first determined the major integrins expressed on the hESCs to reveal the preference of the hESCs for rhLMs, and found that the hESCs mainly expressed integrin {alpha}6{beta}1, which binds predominantly to laminin-111, -332 and -511/-521. When the hESCs were seeded onto rhLMs, the cells indeed adhered markedly to rhLM-332, and to rhLM-511 and rhLM-111 to a lesser extent. The hESCs proliferated on these three rhLMs for several passages while preserving their pluripotency. These results show that rhLM-111, -332, and -511 are good substrates to expand undifferentiated hESCs due to their high affinity to integrin {alpha}6{beta}1 expressed on hESCs.

  3. Salicylic acid induces apoptosis in colon carcinoma cells grown in-vitro: Influence of oxygen and salicylic acid concentration

    SciTech Connect

    Zitta, Karina; Meybohm, Patrick; Bein, Berthold; Huang, Ying; Heinrich, Christin; Scholz, Jens; Steinfath, Markus; Albrecht, Martin

    2012-04-15

    In solid tumors the hypoxic environment can promote tumor progression and resistance to therapy. Recently, acetylsalicylic acid a major component of analgesic drugs and its metabolite salicylic acid (SA) have been shown to reduce the risk of colon cancer, but the mechanisms of action remain still unclear. Here we elucidate the effects of physiologically relevant concentrations of SA on colon carcinoma cells (CaCo-2) grown under normoxic and hypoxic conditions. Western blotting, caspase-3/7 apoptosis assays, MTS cell-proliferation assays, LDH cytotoxicity assays and hydrogen peroxide measurements were performed to investigate the effects of 1 and 10 {mu}M SA on CaCo-2 cells grown under normoxic conditions and cells exposed to hypoxia. Under normoxic conditions, SA did not influence cell proliferation or LDH release of CaCo-2 cells. However, caspase-3/7 activity was significantly increased. Under hypoxia, cell proliferation was reduced and LDH release and caspase-3/7 activities were increased. None of these parameters was altered by the addition of SA under hypoxic conditions. Hypoxia increased hydrogen peroxide concentrations 300-fold and SA significantly augmented the release of hydrogen peroxide under normoxic, but not under hypoxic conditions. Phosphorylation of the pro-survival kinases akt and erk1/2 was not changed by SA under hypoxic conditions, whereas under normoxia SA reduced phosphorylation of erk1/2 after 2 hours. We conclude that in colon carcinoma cells effects of SA on apoptosis and cellular signaling are dependent on the availability of oxygen. -- Highlights: Black-Right-Pointing-Pointer Effects of salicylic acid on colon carcinoma cells grown under normoxic and hypoxic conditions Black-Right-Pointing-Pointer Salicylic acid increases caspase-3/7 activity and hydrogen peroxide release under normoxia Black-Right-Pointing-Pointer Salicylic acid decreases pro-survival erk-1/2 phosphorylation under normoxia Black-Right-Pointing-Pointer Salicylic acid does

  4. Comparison of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in hepatoma cells grown in vivo and in vitro.

    PubMed Central

    Beirne, O R; Watson, J A

    1976-01-01

    Unlike the normal liver, numerous transplantable rodent and human hepatomas are unable to alter their rate of sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-GoA) reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] activity in response to a dietary cholesterol challenge. It has been suggested that this metabolic defect is linked to the process of malignant transformation. Hepatoma 7288C "lacks" feedback regulation of cholesterol synthesis when grown in vivo but expresses this regulatory property when grown in vitro (then called HTC). Therefore, it was used as a model system to answer whether an established hepatoma cell line that modulates its rate of cholesterol synthesis in vitro can express this property when grown in vivo, and whether cells reisolated from the tumor mass have the same regulatory phenotype as before transplantation. Our results show that long-term growth of hepatoma 7288C in tissue culture has not caused a biotransformation that permits feedback regulation of HMG-CoA reductase when the cells are transplanted back into host animals. In addition, HTC cells reisolated from the tumor mass and established in tissue culture continue to have the ability to regulate HMG-CoA reductase activity. Therefore, malignant transformation is not categorically linked to the loss of the cellular components necessary to regulate sterol synthesis and HMG-CoA reductase activity. Images PMID:183207

  5. Autophagic response to cell culture stress in pluripotent stem cells.

    PubMed

    Gregory, Siân; Swamy, Sushma; Hewitt, Zoe; Wood, Andrew; Weightman, Richard; Moore, Harry

    2016-05-01

    Autophagy is an important conserved cellular process, both constitutively as a recycling pathway for long lived proteins and as an upregulated stress response. Recent findings suggest a fundamental role for autophagic processes in the maintenance of pluripotent stem cell function. In human embryonic stem cells (hESCS), autophagy was investigated by transfection of LC3-GFP to visualize autophagosomes and with an antibody to LC3B protein. The presence of the primary cilium (PC) in hESCs as the site of recruitment of autophagy-related proteins was also assessed. HESCs (mShef11) in vitro displayed basal autophagy which was upregulated in response to deprivation of culture medium replacement. Significantly higher levels of autophagy were exhibited on spontaneous differentiation of hESCs in vitro. The PC was confirmed to be present in hESCs and therefore may serve to coordinate autophagy function. PMID:26385182

  6. A new era in the ethics of human embryonic stem cell research.

    PubMed

    Lo, Bernard; Zettler, Patricia; Cedars, Marcelle I; Gates, Elena; Kriegstein, Arnold R; Oberman, Michelle; Reijo Pera, Renee; Wagner, Richard M; Wuerth, Mary T; Wolf, Leslie E; Yamamoto, Keith R

    2005-01-01

    Scientific progress in human embryonic stem cell (hESC) research and increased funding make it imperative to look ahead to the ethical issues generated by the expected use of hESCs for transplantation. Several issues should be addressed now, even though phase I clinical trials of hESC transplantation are still in the future. To minimize the risk of hESC transplantation, donors of materials used to derive hESC lines will need to be recontacted to update their medical history and screening. Because of privacy concerns, such recontact needs to be discussed and agreed to at the time of donation, before new hESC lines are derived. Informed consent for phase I clinical trials of hESC transplantation also raises ethical concerns. In previous phase I trials of highly innovative interventions, allegations that trial participants had not really understood the risk and benefits caused delays in subsequent trials. Thus, researchers should consider what information needs to be discussed during the consent process for hESC clinical trials and how to verify that participants have a realistic understanding of the study. Lack of attention to the special ethical concerns raised by clinical trials of hESC transplantation and their implications for the derivation of new hESC lines may undermine or delay progress toward stem cell therapies.

  7. Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation.

    PubMed

    Benning, C; Huang, Z H; Gage, D A

    1995-02-20

    Cells of the photosynthetic bacterium Rhodobacter sphaeroides grown under phosphate-limiting conditions accumulated nonphosphorous glycolipids and lipids carrying head groups derived from amino acids. Concomitantly, the relative amount of phosphoglycerolipids decreased from 90 to 22 mol% of total polar lipids in the membranes. Two lipids, not detectable in cells grown under standard conditions, were synthesized during phosphate-limited growth. Fast atom bombardment mass spectroscopy, exact mass measurements, 1H NMR spectroscopy, sugar composition analysis, and methylation analysis of the predominant glycolipid led to the identification of the novel compound 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1-->4)-O-beta-D-galactopyr anosyl]glycerol. The second lipid was identified as the betaine lipid 1,2-di-O-acyl-[4'-(N,N,N-trimethyl)-homoserine]glycerol by cochromatography employing an authentic standard from Chlamydomonas reinhardtii, fast atom bombardment mass spectroscopy, exact mass measurements, and 1H NMR spectroscopy. Prior to this observation, the occurrence of this lipid was thought to be restricted to lower plants and algae. Apparently, these newly synthesized nonphosphorous lipids, in addition to the sulfo- and the ornithine lipid also found in R. sphaeroides grown under optimal conditions, take over the role of phosphoglycerolipids in phosphate-deprived cells. PMID:7872771

  8. Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation.

    PubMed

    Benning, C; Huang, Z H; Gage, D A

    1995-02-20

    Cells of the photosynthetic bacterium Rhodobacter sphaeroides grown under phosphate-limiting conditions accumulated nonphosphorous glycolipids and lipids carrying head groups derived from amino acids. Concomitantly, the relative amount of phosphoglycerolipids decreased from 90 to 22 mol% of total polar lipids in the membranes. Two lipids, not detectable in cells grown under standard conditions, were synthesized during phosphate-limited growth. Fast atom bombardment mass spectroscopy, exact mass measurements, 1H NMR spectroscopy, sugar composition analysis, and methylation analysis of the predominant glycolipid led to the identification of the novel compound 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1-->4)-O-beta-D-galactopyr anosyl]glycerol. The second lipid was identified as the betaine lipid 1,2-di-O-acyl-[4'-(N,N,N-trimethyl)-homoserine]glycerol by cochromatography employing an authentic standard from Chlamydomonas reinhardtii, fast atom bombardment mass spectroscopy, exact mass measurements, and 1H NMR spectroscopy. Prior to this observation, the occurrence of this lipid was thought to be restricted to lower plants and algae. Apparently, these newly synthesized nonphosphorous lipids, in addition to the sulfo- and the ornithine lipid also found in R. sphaeroides grown under optimal conditions, take over the role of phosphoglycerolipids in phosphate-deprived cells.

  9. Possible pathways linking ploidy level to cell elongation and cuticular function in hypocotyls of dark-grown Arabidopsis seedlings

    PubMed Central

    Narukawa, Hideki; Yokoyama, Ryusuke; Nishitani, Kazuhiko

    2016-01-01

    abstract The mechanisms underlying correlations between ploidy level and cell size in eukaryotes remain unclear. Recently, we showed that cell length was higher in tetraploid than in diploid dark-grown Arabidopsis hypocotyls. Cuticular function was aberrant, and expression of genes of cuticle formation was reduced. Here, the links between cell elongation, cuticular function, and ploidy level in the etiolated hypocotyl were examined. Seedlings defective in cuticle formation exhibited shorter hypocotyls. This was due to inhibition of cell elongation rather than cell proliferation, indicating that the reduced cuticular function was a consequence of tetraploidy-induced cell elongation rather than its cause. Inhibition of hypocotyl elongation by impaired cuticles was lower in tetraploid than diploid, indicating that tetraploid hypocotyls were less sensitive to cuticular damage. PMID:26618780

  10. Derivation and characterization of human embryonic stem cells on human amnion epithelial cells.

    PubMed

    Lai, Dongmei; Wang, Yongwei; Sun, Jian; Chen, Yifei; Li, Ting; Wu, Yi; Guo, Lihe; Wei, Chunsheng

    2015-05-07

    Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs.

  11. Derivation and characterization of human embryonic stem cells on human amnion epithelial cells

    PubMed Central

    Lai, Dongmei; Wang, Yongwei; Sun, Jian; Chen, Yifei; Li, Ting; Wu, Yi; Guo, Lihe; Wei, Chunsheng

    2015-01-01

    Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs. PMID:25950719

  12. Derivation and characterization of human embryonic stem cells on human amnion epithelial cells.

    PubMed

    Lai, Dongmei; Wang, Yongwei; Sun, Jian; Chen, Yifei; Li, Ting; Wu, Yi; Guo, Lihe; Wei, Chunsheng

    2015-01-01

    Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs. PMID:25950719

  13. Alteration of photochemistry and protein degradation of photosystem II from Chlamydomonas reinhardtii under high salt grown cells.

    PubMed

    Neelam, Satyabala; Subramanyam, Rajagopal

    2013-07-01

    In this study, we evaluated the inhibitory effect of NaCl on cell growth, photochemistry and protein profile of photosystem (PS) II in Chlamydomonas reinhardtii. To study the effect of NaCl on the photosynthetic apparatus, the C. reinhardtii cells were grown at different concentrations (0, 50, 100 and 150 mM). NaCl induced flagellar resorption due to which the cells lost their motility, formation of palmelloids, reduced cell size and slower cell division. Chlorophyll fluorescence transients at different NaCl concentrations had decreased intensities of all peaks (OJIP) indicating the apparent inactivation energies of both donor and acceptor side of PSII. Consequently, inhibition of electron transport occurred particularly at PSII. Further, low temperature emission spectra showed that the rate of damage to the PSII was more when compared to PSI. Also, we have carried out the visible circular dichroism spectra from thylakoids where the major peaks contributed to chlorophyll a and b are equally reduced in different salt grown cells, which may explain the changes at the level of inter pigment-pigment interactions. Furthermore protein profile analysis of PSII revealed that the major subunit of light harvesting complex (LHC)II is more prone to salt stress than core proteins of PSII indicating the light harvesting funnel from LHCII to PSII core is impaired.

  14. Growth and characterization of Czochralski-grown n and p-type GaAs for space solar cell substrates

    NASA Technical Reports Server (NTRS)

    Chen, R. T.

    1983-01-01

    Progress in LEC (liquid encapsulated Czochralski) crystal growth techniques for producing high-quality, 3-inch-diameter, n- and p-type GaAs crystals suitable for solar cell applications is described. The LEC crystals with low dislocation densities and background impurities, high electrical mobilities, good dopant uniformity, and long diffusion lengths were reproducibly grown through control of the material synthesis, growth and doping conditions. The capability for producing these large-area, high-quality substrates should positively impact the manufacturability of highly efficiency, low cost, radiation-hard GaAs solar cells.

  15. Estimating the cell density and invasive radius of three-dimensional glioblastoma tumor spheroids grown in vitro

    NASA Astrophysics Data System (ADS)

    Stein, Andrew M.; Nowicki, Michal O.; Demuth, Tim; Berens, Michael E.; Lawler, Sean E.; Chiocca, E. Antonio; Sander, Leonard M.

    2007-08-01

    To gain insight into brain tumor invasion, experiments are conducted on multicellular tumor spheroids grown in collagen gel. Typically, a radius of invasion is reported, which is obtained by human measurement. We present a simple, heuristic algorithm for automated invasive radii estimation (AIRE) that uses local fluctuations of the image intensity. We then derive an analytical expression relating the image graininess to the cell density for a model imaging system. The result agrees with the experiment up to a multiplicative constant and thus describes a novel method for estimating the cell density from bright-field images.

  16. Cadmium chloride alters mRNA levels of angiogenesis related genes in primary human endometrial endothelial cells grown in vitro.

    PubMed

    Helmestam, Malin; Stavreus-Evers, Anneli; Olovsson, Matts

    2010-11-01

    Cadmium, is known to cause adverse reproductive effects, and classified as an endocrine disrupting chemical (EDC). Human endometrial endothelial cells (HEEC) have a key role in the regulation of endometrial angiogenesis. These cells are known to express estrogen receptors, a feature that makes them potential targets for EDCs such as cadmium. We have designed a co-culture system, in which HEEC were grown in the same cell culture medium as endometrial stromal cells but in separate, communicating chambers. With quantitative PCR, we investigated changes in mRNA expression of genes associated with angiogenesis, sex steroids and endothelial cell specific functions. We found that cadmium altered the mRNA expression of the two important angiogenic molecules VEGF-A and PLGF. Cadmium might thus affect endometrial angiogenesis and as a consequence cause endometrial dysfunction with an increased risk for fertility problems. PMID:20580663

  17. Epitaxial Crystal Silicon Absorber Layers and Solar Cells Grown at 1.8 Microns per Minute: Preprint

    SciTech Connect

    Bobela, D. C.; Teplin, C. W.; Young, D. L.; Branz, H. M.; Stradins, P.

    2011-07-01

    We have grown device-quality epitaxial silicon thin films at growth rates up to 1.8 μm/min, using hot-wire chemical vapor deposition from silane at substrate temperatures below 750 degrees C. At these rates, which are more than 30 times faster than those used by the amorphous and nanocrystalline Si industry, capital costs for large-scale solar cell production would be dramatically reduced, even for cell absorber layers up to 10 ?m thick. We achieved high growth rates by optimizing the three key parameters: silane flow, depletion, and filament geometry, based on our model developed earlier. Hydrogen coverage of the filament surface likely limits silane decomposition and growth rate at high system pressures. No considerable deterioration in PV device performance is observed when grown at high rate, provided that the epitaxial growth is initiated at low rate. A simple mesa device structure (wafer/epi Si/a-Si(i)/a-Si:H(p)/ITO) with a 2.3 um epitaxial silicon absorber layer was grown at 700 nm/min. The finished device had an open-circuit voltage of 0.424 V without hydrogenation treatment.

  18. Improving survival of disassociated human embryonic stem cells by mechanical stimulation using acoustic tweezing cytometry.

    PubMed

    Chen, Di; Sun, Yubing; Deng, Cheri X; Fu, Jianping

    2015-03-24

    Dissociation-induced apoptosis of human embryonic stem cells (hESCs) hampers their large-scale culture. Herein we leveraged the mechanosensitivity of hESCs and employed, to our knowledge, a novel technique, acoustic tweezing cytometry (ATC), for subcellular mechanical stimulation of disassociated single hESCs to improve their survival. By acoustically actuating integrin-bound microbubbles (MBs) to live cells, ATC increased the survival rate and cloning efficiency of hESCs by threefold. A positive correlation was observed between the increased hESC survival rate and total accumulative displacement of integrin-anchored MBs during ATC stimulation. ATC may serve as a promising biocompatible tool to improve hESC culture.

  19. Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells.

    PubMed

    Svensson, Bengt; Nagubothu, Srinivasa R; Nord, Christoffer; Cedervall, Jessica; Hultman, Isabell; Ährlund-Richter, Lars; Tolf, Anna; Hertegård, Stellan

    2015-01-01

    We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p ≤ 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p ≤ 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives. PMID:26557696

  20. Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells

    PubMed Central

    Svensson, Bengt; Nagubothu, Srinivasa R.; Nord, Christoffer; Cedervall, Jessica; Hultman, Isabell; Ährlund-Richter, Lars; Tolf, Anna; Hertegård, Stellan

    2015-01-01

    We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p ≤ 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p ≤ 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives. PMID:26557696

  1. Scalable Culture and Cryopreservation of Human Embryonic Stem Cells on Microcarriers

    PubMed Central

    Nie, Ying; Bergendahl, Veit; Hei, Derek J.; Jones, Jeffrey M.; Palecek, Sean P.

    2009-01-01

    As a result of their pluripotency and potential for unlimited self-renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large-scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor-intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel-coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF-microcarriers was less than that on MEF-plates, the doubling time of hESCs on Matrigel-microcarriers was indistinguishable from that of hESCs expanded on Matrigel-coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier-based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. PMID:19197994

  2. Single Junction InGaP/GaAs Solar Cells Grown on Si Substrates using SiGe Buffer Layers

    NASA Technical Reports Server (NTRS)

    Ringel, S. A.; Carlin, J. A.; Andre, C. L.; Hudait, M. K.; Gonzalez, M.; Wilt, D. M.; Clark, E. B.; Jenkins, P.; Scheiman, D.; Allerman, A.

    2002-01-01

    Single junction InGaP/GaAs solar cells displaying high efficiency and record high open circuit voltage values have been grown by metalorganic chemical vapor deposition on Ge/graded SiGe/Si substrates. Open circuit voltages as high as 980 mV under AM0 conditions have been verified to result from a single GaAs junction, with no evidence of Ge-related sub-cell photoresponse. Current AM0 efficiencies of close to 16% have been measured for a large number of small area cells, whose performance is limited by non-fundamental current losses due to significant surface reflection resulting from greater than 10% front surface metal coverage and wafer handling during the growth sequence for these prototype cells. It is shown that at the material quality currently achieved for GaAs grown on Ge/SiGe/Si substrates, namely a 10 nanosecond minority carrier lifetime that results from complete elimination of anti-phase domains and maintaining a threading dislocation density of approximately 8 x 10(exp 5) per square centimeter, 19-20% AM0 single junction GaAs cells are imminent. Experiments show that the high performance is not degraded for larger area cells, with identical open circuit voltages and higher short circuit current (due to reduced front metal coverage) values being demonstrated, indicating that large area scaling is possible in the near term. Comparison to a simple model indicates that the voltage output of these GaAs on Si cells follows ideal behavior expected for lattice mismatched devices, demonstrating that unaccounted for defects and issues that have plagued other methods to epitaxially integrate III-V cells with Si are resolved using SiGe buffers and proper GaAs nucleation methods. These early results already show the enormous and realistic potential of the virtual SiGe substrate approach for generating high efficiency, lightweight and strong III-V solar cells.

  3. Effects of growth temperature and device structure on GaP solar cells grown by molecular beam epitaxy

    SciTech Connect

    Vaisman, M.; Tomasulo, S.; Masuda, T.; Lang, J. R.; Faucher, J.; Lee, M. L.

    2015-02-09

    Gallium phosphide (GaP) is an attractive candidate for wide-bandgap solar cell applications, possessing the largest bandgap of the III-arsenide/phosphides without aluminum. However, GaP cells to date have exhibited poor internal quantum efficiency (IQE), even for photons absorbed by direct transitions, motivating improvements in material quality and device structure. In this work, we investigated GaP solar cells grown by molecular beam epitaxy over a range of substrate temperatures, employing a much thinner emitter than in prior work. Higher growth temperatures yielded the best solar cell characteristics, indicative of increased diffusion lengths. Furthermore, the inclusion of an AlGaP window layer improved both open-circuit voltage and short wavelength IQE.

  4. Beta 1 integrin binding plays a role in the constant traction force generation in response to varying stiffness for cells grown on mature cardiac extracellular matrix.

    PubMed

    Gershlak, Joshua R; Black, Lauren D

    2015-01-15

    We have previously reported a unique response of traction force generation for cells grown on mature cardiac ECM, where traction force was constant over a range of stiffnesses. In this study we sought to further investigate the role of the complex mixture of ECM on this response and assess the potential mechanism behind it. Using traction force microscopy, we measured cellular traction forces and stresses for mesenchymal stem cells (MSCs) grown on polyacrylamide gels at a range of stiffnesses (9, 25, or 48 kPa) containing either adult rat heart ECM, different singular ECM proteins including collagen I, fibronectin, and laminin, or ECM mimics comprised of varying amounts of collagen I, fibronectin, and laminin. We also measured the expression of integrins on these different substrates as well as probed for β1 integrin binding. There was no significant change in traction force generation for cells grown on the adult ECM, as previously reported, whereas cells grown on singular ECM protein substrates had increased traction force generation with an increase in substrate stiffness. Cells grown on ECM mimics containing collagen I, fibronectin and laminin were found to be reminiscent of the traction forces generated by cells grown on native ECM. Integrin expression generally increased with increasing stiffness except for the β1 integrin, potentially implicating it as playing a role in the response to adult cardiac ECM. We inhibited binding through the β1 integrin on cells grown on the adult ECM and found that the inhibition of β1 binding led to a return to the typical response of increasing traction force generation with increasing stiffness. Our data demonstrates that cells grown on the mature cardiac ECM are able to circumvent typical stiffness related cellular behaviors, likely through β1 integrin binding to the complex composition.

  5. Human ES and iPS cells as cell sources for the treatment of Parkinson's disease: current state and problems.

    PubMed

    Hwang, Dong-Youn; Kim, Dae-Sung; Kim, Dong-Wook

    2010-02-01

    Cell therapy using human embryonic stem cells (hESCs) is a promising therapeutic option for Parkinson's disease (PD), an incurable neurodegenerative disease. A prerequisite for clinical application of hESCs for PD is an efficient and strict differentiation of hESCs into midbrain dopamine (mDA) neuron-like cells, which would be directly translated into high effectiveness of the therapy with minimum risk of undesirable side effects. Due to fruitful efforts from many laboratories, a variety of strategies for improving efficiency of dopaminergic differentiation from hESCs have been developed, mostly by optimizing culture conditions, genetic modification, and modulating intracellular signaling pathways. The rapid advances in the fields of dopaminergic differentiation of hESCs, prevention of tumor formation, and establishment of safe human induced pluripotent stem cells (hiPSCs) would open the door to highly effective, tumor-free, and immune rejection-free cell therapy for PD in the near future.

  6. Cu(In,Ga)Se 2 thin-film solar cells grown with cracked selenium

    NASA Astrophysics Data System (ADS)

    Kawamura, Masahiro; Fujita, Toshiyuki; Yamada, Akira; Konagai, Makoto

    2009-01-01

    Cu(In 1-xGa x)Se 2 (CIGS) films have been grown by using cracked selenium. In conventional evaporation system, the Se atoms were supplied as large clusters (Se x, x>5). However, the size of clusters can be reduced by the thermal cracking. The film qualities grown with small clusters (Se x, x<4) would be improved, since the smaller size molecules easily react with elemental metals, resulting in the reduction of selenium vacancies and the enhancement of surface migration. The CIGS films were deposited by the three-stage method with cracked selenium, and the films were evaluated by SEM, XRD, EDX, C- V measurement and admittance spectroscopy. It was found from the C- V characteristics that the carrier concentrations of the CIGS films grown with cracked selenium were increased with increasing the cracking temperature. The result clearly showed that the use of cracked selenium was effective for reduction of selenium vacancies. The conversion efficiency of 15.4% was obtained by using cracked selenium at a cracking temperature of 500 °C.

  7. Single-crystal p-i-n-Si thin-film solar cells grown on Si substrate by sputter epitaxy

    NASA Astrophysics Data System (ADS)

    Yeh, Wenchang; Tatebe, Kyohei

    2015-08-01

    An intrinsic sputter-epitaxial (SE) Si film with a thickness of 1000 nm and a 50-nm-thick n+ SE-Si film were successfully grown as the light-absorbing layer and emitter layer, respectively, on a heavily doped p-Si(100) wafer to form the p-i-n junction of a solar cell (SC). Heavily doped n+ SE-Si with an electron concentration n of 3 × 1020 cm-3 was grown by cosputtering of Sb with Si. The characteristics of SE-Si grown at 310 °C was investigated in relation to annealing temperature. The oxygen concentration in SE-Si was ˜1018 cm-3, which was found to originate from the gas released in the chamber. Oxygen-induced thermal donors then became the source of n in the film, and n was reduced to 1 × 1016 cm-3 after forming-gas annealing at 700 °C because the thermal donors were neutralized by hydrogen. The SC exhibited a maximum internal quantum efficiency of 73.7%.

  8. Adipose-derived stromal cells grown on a hydroxyapatite scaffold can support hematopoiesis in regenerated bone marrow in vivo.

    PubMed

    Ueda, Takahiro; Fujita, Atsushi; Ogawa, Rei; Itoh, Yasuhiko; Fukunaga, Yoshitaka; Shimada, Takashi; Migita, Makoto

    2014-06-01

    Osteoblastic cells are a key component of the bone marrow (BM) stem cell niche and help regulate hematopoietic stem cells (HSCs). We have previously demonstrated that adipose-derived stromal cells (ADSCs) can differentiate into both osteogenic and chondrogenic cells in vitro. The current study examined whether the anatomical architecture of the BM could be regenerated in vivo by using ADSCs cultured on a hydroxyapatite (HA) scaffold. ADSCs from GFP transgenic mice were cultured in vitro on an HA scaffold. The scaffold with the attached cells was implanted subcutaneously onto the backs of C57/BL6 (Ly5.2) recipient mice. Lineage-negative (Lin-) Ly5.1 BM cells transduced with a lentiviral vector containing the luciferase (Luc) gene were intravenously administered to the recipient mice after lethal irradiation. Eight weeks after BM transplantation, the scaffolds were removed from the first recipient mice and subcutaneously implanted into lethally irradiated second recipient mice. The biodistribution and kinetics of Luc(+) Ly5.1 cells were monitored by bioluminescence imaging and flow cytometry. Luc(+) hematopoietic cells were present in the scaffolds of the secondary implanted mice for at least 8 months. Subcutaneous injection of G-CSF resulted in wide distribution of bioluminescence signals from the original scaffolds to the whole body. Therefore, BM regenerated using ADSCs grown on an HA scaffold can support HSC populations in vivo, suggesting that a functional BM niche is reconstituted. These results may have a significant impact on the development of therapeutic strategies for various hematopoietic diseases.

  9. Cytotoxicity and mutagenicity of vapor-phase pollutants in rat lung epithelial cells and Chinese hamster ovary cells grown on collagen gels

    SciTech Connect

    Zamora, P.O.; Benson, J.M.; Marshall, T.C.; Mokler, B.V.; Li, A.P.; Dahl, A.R.; Brooks, A.L.; McClellan, R.O.

    1983-01-01

    Lung epithelial cell (cell line designated LEC) and Chinese hamster ovary (CHO) cells were grown on hydrated collagen gels and exposed directly to toxic vapor-phase pollutants. The cells were exposed to graded concentrations of phenol, formaldehyde, a volatile fraction of process stream material from an experimental coal gasifier, and the nonparticulate, vapor phase of diesel engine exhaust. During exposures, the cells were maintained at an air/collagen interface by removing the medium overlying the hydrated collagen gel. Morphological changes indicative of cell retraction were found in LEC cell cultures exposed to phenol, formaldehyde, or diesel exhaust. Damage following exposure to the toxicants was quantitated in LEC and CHO cells by Trypan blue dye exclusion, a measure of plasma membrane integrity. Clone-forming ability was also used to measure cell survival in CHO cells. When measured by Trypan blue dye exclusion, phenol (EC50 = 2.1 mg/l) caused membrane damage to LEC cells but not CHO cells, while formaldehyde (EC50 = 31 and 42 ..mu..g/l for LEC and CHO, respectively) and diesel exhaust (EC50 = 11 and 29% of tailpipe exhaust in LEC and CHO cells, respectively) caused damage to both cell types. No cytotoxicity was observed in LEC or CHO cells exposed to the fraction from the coal gasifier. Essentially no mutagenic activity was associated with the exposure of CHO cells to formaldehyde or the vapor phase of diesel exhaust. Mutagenic activity was found in CHO cells exposed to ethylene oxide, the positive control.

  10. Pemetrexed alters folate phenotype and inflammatory profile in EA.hy 926 cells grown under low-folate conditions

    PubMed Central

    Hammons, Andrea L.; Summers, Carolyn M.; Jochems, Jeanine; Arora, Jasbir S.; Zhang, Suhong; Blair, Ian A.; Whitehead, Alexander S.

    2014-01-01

    Elevated homocysteine is a risk marker for several major human pathologies. Emerging evidence suggests that perturbations of folate/homocysteine metabolism can directly modify production of inflammatory mediators. Pemetrexed acts by inhibiting thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). EA.hy 926 cells grown under low (“Lo”) and high (“Hi”) folate conditions were treated with pemetrexed. The concentrations of several intracellular folate derivatives were measured using LC-MRM/MS. Lo cells had lower total folate concentrations and a different distribution of the intracellular folate derivatives than Hi cells. Treatment with pemetrexed caused a decrease in individual folate analytes. Microarray analysis showed that several genes were significantly up or down-regulated in pemetrexed treated Lo cells. Several of the significantly up-regulated transcripts were inflammatory. Changes in transcript levels of selected targets, including C3, IL-8, and DHFR, were confirmed by quantitative RT-PCR. C3 and IL-8 transcript levels were increased in pemetrexed-treated Lo cells relative to Lo controls; DHFR transcript levels were decreased. In Lo cells, IL-8 and C3 protein concentrations were increased following pemetrexed treatment. Pemetrexed drug treatment was shown in this study to have effects that lead to an increase in pro-inflammatory mediators in Lo cells. No such changes were observed in Hi cells, suggesting that pemetrexed could not modify the inflammatory profile in the context of cellular folate sufficiency. PMID:22975265

  11. Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface.

    PubMed

    Lee, Yung; Dizzell, Sara E; Leung, Vivian; Nazli, Aisha; Zahoor, Muhammad A; Fichorova, Raina N; Kaushic, Charu

    2016-01-01

    The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions. PMID:27589787

  12. Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface

    PubMed Central

    Lee, Yung; Dizzell, Sara E.; Leung, Vivian; Nazli, Aisha; Zahoor, Muhammad A.; Fichorova, Raina N.; Kaushic, Charu

    2016-01-01

    The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions. PMID:27589787

  13. "allometry" Deterministic Approaches in Cell Size, Cell Number and Crude Fiber Content Related to the Physical Quality of Kangkong (Ipomoea reptans) Grown Under Different Plant Density Pressures

    NASA Astrophysics Data System (ADS)

    Selamat, A.; Atiman, S. A.; Puteh, A.; Abdullah, N. A. P.; Mohamed, M. T. M.; Zulkeefli, A. A.; Othman, S.

    Kangkong, especially the upland type (Ipomoea reptans) is popularly consumed as a vegetable dish in the South East Asian countries for its quality related to Vitamins (A and C) and crude fiber contents. Higher fiber contents would prevent from the occurrence of colon cancer and diverticular disease. With young stem edible portion, its cell number and size contribute to the stem crude fiber content. The mathematical approach of allometry of cell size, number, and fiber content of stem could be used in determining the 'best' plant density pressure in producing the quality young stem to be consumed. Basically, allometry is the ratio of relative increment (growth or change) rates of two parameters, or the change rate associated to the log of measured variables relationship. Kangkog grown equal or lower than 55 plants m-2 produced bigger individual plant and good quality (physical) kangkong leafy vegetable, but with lower total yield per unit area as compared to those grown at higher densities.

  14. Separation of SSEA-4 and TRA-1-60 labelled undifferentiated human embryonic stem cells from a heterogeneous cell population using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS).

    PubMed

    Fong, Chui Yee; Peh, Gary S L; Gauthaman, Kalamegam; Bongso, Ariff

    2009-03-01

    A major concern in human embryonic stem cell (hESC)-derived cell replacement therapy is the risk of tumorigenesis from undifferentiated hESCs residing in the population of hESC-derived cells. Separation of these undifferentiated hESCs from the differentiated derivatives using cell sorting methods may be a plausible approach in overcoming this problem. We therefore explored magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) to separate labelled undifferentiated hESCs from a heterogeneous population of hESCs and hepatocellular carcinoma cells (HepG2) deliberately mixed respectively at different ratios (10:90, 20:80, 30:70, 40:60 and 50:50) to mimic a standard in vitro differentiation protocol, instead of using a hESC-differentiated cell population, so that we could be sure of the actual number of cells separated. HES-3 and HES-4 cells were labelled in separate experiments for the stem cell markers SSEA-4 and TRA-1-60 using primary antibodies. Anti-PE magnetic microbeads that recognize the PE-conjugated SSEA-4 labelled hESCs was added to the heterogeneous cell mixture and passed through the MACS column. The cells that passed through the column ('flow-through' fraction) and those retained ('labelled' fraction') were subsequently analysed using FACS. The maximum efficacy of hESCs retention using MACS was 81.0 +/- 2.9% (HES-3) and 83.6 +/- 4.2% (HES-4). Using FACS, all the undifferentiated hESCs labelled with the two cell-surface markers could be removed by selective gating. Both hESCs and HepG2 cells in the 'flow-through' fraction following MACS separation were viable in culture whereas by FACS separation only the HepG2 cells were viable. FACS efficiently helps to eliminate the undifferentiated hESCs based on their cell-surface antigens expressed.

  15. Expand and Regularize Federal Funding for Human Pluripotent Stem Cell Research

    ERIC Educational Resources Information Center

    Owen-Smith, Jason; Scott, Christopher Thomas; McCormick, Jennifer B.

    2012-01-01

    Human embryonic stem cell (hESC) research has sparked incredible scientific and public excitement, as well as significant controversy. hESCs are pluripotent, which means, in theory, that they can be differentiated into any type of cell found in the human body. Thus, they evoke great enthusiasm about potential clinical applications. They are…

  16. Temperature coefficients and radiation induced DLTS spectra of MOCVD grown n(+)p InP solar cells

    NASA Technical Reports Server (NTRS)

    Walters, Robert J.; Statler, Richard L.; Summers, Geoffrey P.

    1991-01-01

    The effects of temperature and radiation on n(+)p InP solar cells and mesa diodes grown by metallorganic chemical vapor deposition (MOCVD) were studied. It was shown that MOCVD is capable of consistently producing good quality InP solar cells with Eff greater than 19 percent which display excellent radiation resistance due to minority carrier injection and thermal annealing. It was also shown that universal predictions of InP device performance based on measurements of a small group of test samples can be expected to be quite accurate, and that the degradation of an InP device due to any incident particle spectrum should be predictable from a measurement following a single low energy proton irradiation.

  17. Development of a 2.0 eV AlGaInP Solar Cell Grown by OMVPE

    SciTech Connect

    Perl, Emmett E.; Simon, John; Geisz, John F.; Olavarria, Waldo; Young, Michelle; Duda, Anna; Dippo, Pat; Friedman, Daniel J.; Steiner, Myles A.

    2015-06-14

    AlGaInP solar cells with a bandgap (Eg) of ~2.0 eV are developed for use in next-generation multijunction photovoltaic devices. This material system is of great interest for both space and concentrator photovoltaics due to its high bandgap, which enables the development of high-efficiency five-junction and six-junction devices and is also useful for solar cells operated at elevated temperatures. In this work, we explore the conditions for the Organometallic Vapor Phase Epitaxy (OMVPE) growth of AlGaInP and study their effects on cell performance. A ~2.0 eV AlGaInP solar cell is demonstrated with an open circuit voltage (VOC) of 1.59V, a bandgap-voltage offset (WOC) of 420mV, a fill factor (FF) of 88.0%, and an efficiency of 14.8%. These AlGaInP cells have attained a similar FF, WOC and internal quantum efficiency (IQE) to the best upright GaInP cells grown in our lab to date.

  18. Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

    NASA Astrophysics Data System (ADS)

    Stringari, Chiara; Sierra, Robert; Donovan, Peter J.; Gratton, Enrico

    2012-04-01

    We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation.

  19. Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

    PubMed Central

    Stringari, Chiara; Sierra, Robert; Donovan, Peter J.

    2012-01-01

    Abstract. We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation. PMID:22559690

  20. Triploid human embryonic stem cells derived from tripronuclear zygotes displayed pluripotency and trophoblast differentiation ability similar to the diploid human embryonic stem cells.

    PubMed

    Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Virutamasen, Pramuan; Pruksananonda, Kamthorn

    2016-04-22

    Because the diploid human embryonic stem cells (hESCs) can be successfully derived from tripronuclear zygotes thus, they can serve as an alternative source of derivation of normal karyotype hESC lines. The aim of the present study was to compare the pluripotency and trophoblast differentiation ability of hESCs derived from tripronuclear zygotes and diploid hESCs. In the present study, a total of 20 tripronuclear zygotes were cultured; 8 zygotes developed to the blastocyst stage and 1 hESC line was generated. Unlike the previous studies, chromosomal correction of tripronuclear zygotes during derivation of hESCs did not occur. The established line carries 3 sets of chromosomes and showed a numerical aberration. Although the cell line displayed an abnormal chromosome number, it was found the cell line has been shown to be pluripotent with the ability to differentiate into 3 embryonic germ layers both in vitro and in vivo. The expression of X inactive specific transcript (XIST) in mid-passage (passage 42) of undifferentiated triploid hESCs was detected, indicating X chromosome inactivation of the cell line. Moreover, when this cell line was induced to differentiate toward the trophoblast lineage, morphological and functional trophoblast cells were observed, similar to the diploid hESC line. PMID:26821869

  1. Epitaxially Grown Collagen Fibrils Reveal Diversity in Contact Guidance Behavior among Cancer Cells

    PubMed Central

    2015-01-01

    Invasion of cancer cells into the surrounding tissue is an important step during cancer progression and is driven by cell migration. Cell migration can be random, but often it is directed by various cues such as aligned fibers composed of extracellular matrix (ECM), a process called contact guidance. During contact guidance, aligned fibers bias migration along the long axis of the fibers. These aligned fibers of ECM are commonly composed of type I collagen, an abundant structural protein around tumors. In this paper, we epitaxially grew several different patterns of organized type I collagen on mica and compared the morphology and contact guidance behavior of two invasive breast cancer cell lines (MDA-MB-231 and MTLn3 cells). Others have shown that these cells randomly migrate in qualitatively different ways. MDA-MB-231 cells exert large traction forces, tightly adhere to the ECM, and migrate with spindle-shaped morphology and thus adopt a mesenchymal mode of migration. MTLn3 cells exert small traction forces, loosely adhere to the ECM, and migrate with a more rounded morphology and thus adopt an amoeboid mode of migration. As the degree of alignment of type I collagen fibrils increases, cells become more elongated and engage in more directed contact guidance. MDA-MB-231 cells perceive the directional signal of highly aligned type I collagen fibrils with high fidelity, elongating to large extents and migrating directionally. Interestingly, behavior in MTLn3 cells differs. While highly aligned type I collagen fibril patterns facilitate spreading and random migration of MTLn3 cells, they do not support elongation or directed migration. Thus, different contact guidance cues bias cell migration differently and the fidelity of contact guidance is cell type dependent, suggesting that ECM alignment is a permissive cue for contact guidance, but requires a cell to have certain properties to interpret that cue. PMID:25531276

  2. Enzymatic Detachment of Therapeutic Mesenchymal Stromal Cells Grown on Glass Carriers in a Bioreactor

    PubMed Central

    Salzig, Denise; Schmiermund, Alexandra; P. Grace, Pablo; Elseberg, Christiane; Weber, Christian; Czermak, Peter

    2013-01-01

    Cell therapies require the in vitro expansion of adherent cells such as mesenchymal stromal cells (hMSCs) in bioreactor systems or other culture environments, followed by cell harvest. As hMSCs are strictly adherent cells, cell harvest requires cell detachment. The use of hMSCs for cell therapy requires GMP production in accordance with the guidelines for advanced therapeutic medical products. Therefore, several GMP-conform available proteolytic enzymes were investigated for their ability to promote hMSC detachment. An allogeneic hMSC cell line (hMSC-TERT) that is used in clinical trials in the form of alginate cell capsules was chosen as a model. This study investigated the influence of several factors on the outcome of proteolytic hMSC-TERT detachment. Therefore, hMSC-TERT detachment was analyzed in different cultivation systems (static, dynamic) and in combination with further cell processing including encapsulation. Only two of the commercially available enzymes (AccutaseTM, TrypZeanTM) that fulfill all process requirements (commercial availability, cost, GMP conditions during manufacturing and non-animal origin) are found to be generally suitable for detaching hMSC-TERT. Combining cell detachment with encapsulation demonstrated a high impact of the experimental set up on cell damage. It was preferable to reduce the temperature during detachment and limit the detachment time to a maximum of 20 minutes. Cell detachment in static systems was not comparable with detachment in dynamic systems. Detachment yields in dynamic systems were lower and cell damage was higher for the same experimental conditions. Finally, only TrypZeanTM seemed to be suitable for the detachment of hMSC-TERT from dynamic reactor systems. PMID:24478807

  3. Human amniotic fluid stem cells support undifferentiated propagation and pluripotency of human embryonic stem cell without b-FGF in a density dependent manner.

    PubMed

    Ma, Xiaorong; Li, Huanqi; Xin, Shujia; Ma, Yueting; Ouyang, Tianxiang

    2014-01-01

    Human embryonic stem cells (hESCs) are pluripotent cells which can give rise to almost all adult cell lineages. Culture system of hESCs is complex, requiring exogenous b-FGF and feeder cell layer. Human mesenchymal stem cells (MSCs) not only synthesize soluble cytokines or factors such as b-FGF, but also provide other mechanism which might play positive role on sustaining hESCs propagation and pluripotency. Human amniotic fluid stem (AFS) cells, which share characteristics of both embryonic and adult stem cells, have been regarded as promising cells for regenerative medicine. Taking advantage by AFS cells, we studied the ability of AFS cells in supporting undifferentiated propagation and pluripotency of Chinese population derived X-01 hESCs. Human AF-type amniotic fluid stem cells (hAF-AFSCs) transcribed genes including Activin A, TGF-β1, Noggin and b-FGF, which involved in maintaining pluripotency and self-renewal of hESCs. Compared to mouse embryonic fibroblasts (MEFs), hAF-AFSCs secreted higher concentration of b-FGF which was important in hESCs culture (P < 0.05). The hESCs were propagated more than 30 passages on hAF-AFSCs layer with exogenous b-FGF supplementation, keeping undifferentiated status. While exogenous b-FGF was obviated, propagation of hESCs with undifferentiated status was dependent on density of hAF-AFSC feeder layer. Lower density of hAF-AFSCs resulted in rapid decline in undifferentiated clone number, while higher ones hindered the growth of colonies. The most appropriate hAF-AFSCs feeder density to maintain the X-01 hESC line without exogenous b-FGF was 15-20×10(4)/well. To the best of our knowledge, this is the first study demonstrating that hAF-AFSCs could support undifferentiated propagation and pluripotency of Chinese population derived hESCs without exogenous b-FGF supplementation.

  4. Astrocytes grown in oculo: Expression of cell morphologies on the iris as revealed by GFA immunohistochemistry.

    PubMed

    Björklund, H

    1984-01-01

    Using two experimental approaches, the morphology of central astrocytes growing in vivo with the iris as a substratum were studied. When irides with mature intraocular grafts of cortex cerebri or locus coeruleus were stretch-prepared as whole mounts and processed for immunohistochemistry with antiserum against glial fibrillary acidic protein (GFA), a restricted halo of fluorescent cells and fibers was seen surrounding the grafts. Similarly, injection into the anterior eye chamber of adult rats, of a cell suspension prepared from cortex cerebri of 10-day-old rat pups gave rise to both multiple GFA-positive astrocytic islets of different sizes and cell densities as well as scattered individual cells on the anterior surface of the host iris. In contrast, astrocytes from similar cell suspensions prepared from young adult animals survived very poorly. In both types of experiments, a large variation in cell morphology ranging from immature epitheloid, via large flat cells with few thick processes, to typical mature star-shaped astrocytes was observed. This morphological variation is in agreement with that reported for similar cells in tissue culture. Immature-looking cells always had a strong perinuclear fluorescence; an inverse correlation was observed between cell body size and development of cell processes. Likewise, the fluorescence intensity was higher in well-developed cells as compared to more immature ones. The morphology of individual cells did not seem to be dependent upon the time in oculo, since no difference was observed between GFA-positive cells on irides examined 10 days and 6 weeks after injection of a cell suspension. Similarly, a high number of immature-looking cells was seen in irides with locus coeruleus transplants grafted more than 6 months earlier. Instead, the cell density seemed to be the crucial factor. Thus, star-shaped, well-developed cells were seen growing singly or in less dense groups whereas denser areas contained mainly immature

  5. Astrocytes grown in oculo: Expression of cell morphologies on the iris as revealed by GFA immunohistochemistry.

    PubMed

    Björklund, H

    1984-01-01

    Using two experimental approaches, the morphology of central astrocytes growing in vivo with the iris as a substratum were studied. When irides with mature intraocular grafts of cortex cerebri or locus coeruleus were stretch-prepared as whole mounts and processed for immunohistochemistry with antiserum against glial fibrillary acidic protein (GFA), a restricted halo of fluorescent cells and fibers was seen surrounding the grafts. Similarly, injection into the anterior eye chamber of adult rats, of a cell suspension prepared from cortex cerebri of 10-day-old rat pups gave rise to both multiple GFA-positive astrocytic islets of different sizes and cell densities as well as scattered individual cells on the anterior surface of the host iris. In contrast, astrocytes from similar cell suspensions prepared from young adult animals survived very poorly. In both types of experiments, a large variation in cell morphology ranging from immature epitheloid, via large flat cells with few thick processes, to typical mature star-shaped astrocytes was observed. This morphological variation is in agreement with that reported for similar cells in tissue culture. Immature-looking cells always had a strong perinuclear fluorescence; an inverse correlation was observed between cell body size and development of cell processes. Likewise, the fluorescence intensity was higher in well-developed cells as compared to more immature ones. The morphology of individual cells did not seem to be dependent upon the time in oculo, since no difference was observed between GFA-positive cells on irides examined 10 days and 6 weeks after injection of a cell suspension. Similarly, a high number of immature-looking cells was seen in irides with locus coeruleus transplants grafted more than 6 months earlier. Instead, the cell density seemed to be the crucial factor. Thus, star-shaped, well-developed cells were seen growing singly or in less dense groups whereas denser areas contained mainly immature

  6. Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification.

    PubMed

    Boura, C; Menu, P; Payan, E; Picart, C; Voegel, J C; Muller, S; Stoltz, J F

    2003-09-01

    Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.

  7. Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

    NASA Technical Reports Server (NTRS)

    Hammond, Dianne K.; Becker, Jeanne; Holubec, K.; Baker, T. L.; Love, J. E.

    2004-01-01

    Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LN1) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate Containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered TradeMark)Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark)a software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

  8. Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

    NASA Technical Reports Server (NTRS)

    Hammond, Dianne K.; Becker, Jeanne; Elliott, T. F.; Holubec, K.; Baker, T. L.; Love, J. E.

    2004-01-01

    Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LNI) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered Trademark) Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark) software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

  9. Serum-stimulated cyclic-AMP production in S49 lymphoma cells grown in serum-free medium.

    PubMed

    Darfler, F J; Mullen, M D; Insel, P A

    1984-03-23

    Growth of S49 lymphoma cells with horse serum leads to an increase in cellular cAMP phosphodiesterase activity and a resultant loss of hormone- and cholera-toxin-stimulated cAMP accumulation. We now show that the serum requires protein synthesis to produce these effects. Further, we show that acute addition of serum to wild-type S49 cells, grown in serum-free medium, rapidly (under 2 min) and transiently (under 30 min) stimulates cellular cAMP, 10-fold over basal levels. This 'acute' effect of serum was not observed in UNC S49 cells, suggesting that a functional Ns, the guanine nucleotide regulatory component that mediates stimulation of adenylate cyclase, is required for the serum-mediated stimulation of cellular cAMP. Serum added acutely to wild-type S49 cells also augmented cAMP accumulation in response to isoproterenol and forskolin. The half-maximally effective concentrations of horse serum that acutely stimulated or more slowly decreased the cAMP accumulation were approx. 0.2% and 2.0%, respectively. Preliminary attempts to characterize further the serum factor indicate that it has a high (250 000-300 000) molecular weight and is insensitive to boiling; chromatography on Sepharose CL-6B yields a 100-fold purification. Thus, the serum contains one or more components that activate adenylate cyclase, increase cellular cAMP levels and ultimately induce cAMP phosphodiesterase in S49 lymphoma cells. PMID:6322858

  10. Positioning effects on quantum dot solar cells grown by molecular beam epitaxy

    SciTech Connect

    Zhou, D.; Sharma, G.; Fimland, B. O.; Vullum, P. E.; Thomassen, S. F.; Holmestad, R.; Reenaas, T. W.

    2010-02-22

    We report current-voltage and spectral response characteristics of high density InAs/GaAs quantum dot (QD) solar cells with different positions where dots are located. The short circuit current density (J{sub sc}), open circuit voltage (V{sub oc}), and external quantum efficiency of these cells under air mass 1.5 are presented and compared with a GaAs reference cell. An extended photoresponse in contrast to the GaAs reference cell was confirmed for all these cells. The effect of inserting QD layers into emitter and base region on device performance is shown. The J{sub sc} is reduced, while the V{sub oc} is maintained. The cell with QDs located toward the base side shows better performance, confirmed by both current-voltage and spectral response measurements.

  11. Effect of Hypergravity on Localization Calcium Ions in Plant Cells Grown in Vivo and in Vitro

    NASA Astrophysics Data System (ADS)

    Nedukha, Olena

    Using plant callus tissues and Arabidopsis thaliana plants as model systems we have been investigated the effect of hypergravity on the localization and relative content of calcium ions in photosynthesizing cells. The tobacco callus cells in log stage of growth and mesophyll cells from developed A. thaliana leaves were used in the experiments. Plant samples were exposed to hypergravity at 6.5 g, 10g and 14 g for 15-60 min. After centrifugation, dye Fluo-4 was loaded in the control leaves and the centrifuged samples by the standard cytochemical method. Observation of calcium fluorescence was carried out with a laser confocal microscope LSM 5 Pascal at the excitation wave 488 nm (by the argon laser), at emission wavelength 516 nm. The data of the calcium ion distribution and quantification in cells were obtained using software "Pascal" (Carl Zeiss). The effect of hypergravity on redistribution of calcium ions in plant cells has been established. This effect is depended from exposure time and from the value of hypergravity. The cells cultivated in vitro is showed fast response to hypergravity influence. Plasmolysis cells and calcium domains formation have been observed in most of callus cells. This influence was like to that, which was wrote in Funaria hygrometrica protonema cells after 8.5 g influence (Sytnik et al., 1984). Leaf cells of A. thaliana were of less responsively to hypergravity than callus cells. Sytnik K, Kordyum E, Nedukha O. et al. 1984. Plant Cell Under Change of Geophysical Factors. Kiev: Naukova Dumka, 1-134 p.

  12. Strain-balanced MQW pin solar cells grown using a robot-loading showerhead reactor

    NASA Astrophysics Data System (ADS)

    Roberts, J. S.; Airey, R.; Hill, G.; Calder, C.; Barnham, K. W. J.; Lynch, M.; Tibbits, T.; Johnson, D.; Pakes, A.; Grantham, T.

    2007-01-01

    A touch-screen controlled, robot-loading system for the Thomas Swan 7×2 flip-top showerhead reactor has been developed. The reactor has been configured for the growth of GaAs and InP materials and has been used to prepare strain-balanced MQW (SBMQW) pin solar cell material on GaAs substrates. Both material characterisation and solar cell performance for SBMQW pin cells are described.

  13. Method of measuring nitric oxide release by vascular endothelial cells grown in microfluidic channels

    NASA Astrophysics Data System (ADS)

    Hosseinpour, S.; Liu, A. C.; Barakat, A. I.; Choy, J. C.; Gray, B. L.

    2014-03-01

    In this paper, a simple and versatile method is presented which enables detection of nitric oxide (NO) released from vascular endothelial cells (ECs) cultured in microfluidic structures. The culturing system and NO measurement method allow cell shape to be controlled in a non-invasive manner using microfluidic structures while NO release is monitored for cell shape versus function studies. The culturing system consists of arrays of polydimethylsiloxane (PDMS) fluidic channels 120 micrometers in depth and ranging from 100 micrometers to 3 mm in width. The number of channels in each array is varied to yield a constant cell culture surface area (75 mm2) independent of channel width. The channel surfaces are collagen-coated and ECs are cultured to confluence within the channels. A cell scraper is then used to scrape extraneous cells cultured between channels, and NO measurements are made 18 to 24 hours later. A chemiluminescence-based sensor system (NOA 280i, Sievers NO Analyzer) is utilized to measure sample NO. Initial results indicate that NO concentrations can be measured from different microfluidic channel-containing samples using this method. It is shown that there is no significant difference in NO concentration derived from channels of different widths even though the degree of cell elongation varies due to physical constraint by microfluidic channel walls. However, cells treated with TNFα release more NO than untreated cells in fluidic channels, which is comparable to the function of ECs cultured in conventional culturing systems such as culturing dishes.

  14. The density of apical cells of dark-grown protonemata of the moss Ceratodon purpureus

    NASA Technical Reports Server (NTRS)

    Schwuchow, J. M.; Kern, V. D.; Wagner, T.; Sack, F. D.

    2000-01-01

    Determinations of plant or algal cell density (cell mass divided by volume) have rarely accounted for the extracellular matrix or shrinkage during isolation. Three techniques were used to indirectly estimate the density of intact apical cells from protonemata of the moss Ceratodon purpureus. First, the volume fraction of each cell component was determined by stereology, and published values for component density were used to extrapolate to the entire cell. Second, protonemal tips were immersed in bovine serum albumin solutions of different densities, and then the equilibrium density was corrected for the mass of the cell wall. Third, apical cell protoplasts were centrifuged in low-osmolarity gradients, and values were corrected for shrinkage during protoplast isolation. Values from centrifugation (1.004 to 1.015 g/cm3) were considerably lower than from other methods (1.046 to 1.085 g/cm3). This work appears to provide the first corrected estimates of the density of any plant cell. It also documents a method for the isolation of protoplasts specifically from apical cells of protonemal filaments.

  15. Hall Effect Studies of AlGaAs Grown by Liquid-Phase Epitaxy for Tandem Solar Cell Applications

    NASA Astrophysics Data System (ADS)

    Zhao, Xin; Montgomery, Kyle H.; Woodall, Jerry M.

    2014-11-01

    We report results from Hall effect studies on Al x Ga1- x As ( x = 0.23-0.24) with bandgap energies of 1.76 ± 0.01 eV grown by liquid-phase epitaxy (LPE). Room-temperature Hall measurements on unintentionally doped AlGaAs revealed p-type background doping for concentrations in the range 3.7-5.2 × 1016 cm-3. Sn, Te, Ge, and Zn-doped AlGaAs were also characterized to study the relationship between doping concentrations and the atomic fractions of the dopants in the melt. Temperature-dependent Hall measurements were performed to determine the activation energies of the four dopants. Deep donor levels (DX centers) were dominant for Sn-doped Al0.24Ga0.76As, but not for Te-doped Al0.24Ga0.76As. Comparison of the temperature-dependent Hall effect results for unintentionally and intentionally doped Al0.24Ga0.76As indicated that the impurity contributing to the p-type background doping had the same activation energy as Mg. We thus suggest a Te-doped emitter and an undoped or Ge-doped base to maximize the efficiency of Al x Ga1- x As ( x ˜ 0.23) solar cells grown by LPE.

  16. Quantum yield spectra and I-V properties of a GaAs solar cell grown on a Ge substrate

    NASA Astrophysics Data System (ADS)

    Partain, L. D.; Virshup, G. F.; Kaminar, N. R.

    A measured quantum yield spectrum showed a weak Ge junction in tandem with a strong GaAs junction for a heteroface p/n GaAs junction grown by MOCVD (metal-organic chemical vapor deposition) on an n-Ge substrate. In natural sunlight, this device had a notched-shaped I-V that limited its fill factor (FF) to 0.612 and its efficiency to 18 percent AM2.6D, which are values below those typical of similar heteroface p/n GaAs junctions grown on n-GaAs substrates. The notch disappeared and the FF increased to 0.806 under infrared-rich incandescent light. The notch deepened and the FF decreased to 0.606 under infrared-poor xenon light. This is modeled as a Ge junction with a soft I-V in tandem with a standard GaAs p/n junction. Calculations of the relative infrared content of sunlight predicts that this cell's FF should progressively decrease as the air mass spectrum progressively loses infrared content in going from AM2.6 through AM1.5 to AM0. The best model found for the Ge junction transport properties is an n-GaAs/n-Ge isotype heterojunction possibly controlled by space-charge-limited-current mechanisms.

  17. Gene expression patterns in granulosa cells and oocytes at various stages of follicle development as well as in in vitro grown oocyte-and-granulosa cell complexes.

    PubMed

    Munakata, Yasuhisa; Kawahara-Miki, Ryoka; Shiratsuki, Shogo; Tasaki, Hidetaka; Itami, Nobuhiko; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2016-08-25

    Follicle development is accompanied by proliferation of granulosa cells and increasing oocyte size. To obtain high-quality oocytes in vitro, it is important to understand the processes that occur in oocytes and granulosa cells during follicle development and the differences between in vivo and in vitro follicle development. In the present study, oocytes and granulosa cells were collected from early antral follicles (EAFs, 0.5-0.7 mm in diameter), small antral follicles (SAFs, 1-3 mm in diameter), large antral follicles (LAFs, 3-7 mm in diameter), and in vitro grown oocyte-and-granulosa cell complexes (OGCs), which were cultured for 14 days after collection from EAFs. Gene expression was analyzed comprehensively using the next-generation sequencing technology. We found top upstream regulators during the in vivo follicle development and compared them with those in in vitro developed OGCs. The comparison revealed that HIF1 is among the top regulators during both in vivo and in vitro development of OGCs. In addition, we found that HIF1-mediated upregulation of glycolysis in granulosa cells is important for the growth of OGCs, but the cellular metabolism differs between in vitro and in vivo grown OGCs. Furthermore, on the basis of comparison of upstream regulators between in vivo and in vitro development of OGCs, we believe that low expression levels of FLT1 (VEGFA receptor), SPP1, and PCSK6 can be considered causal factors of the suboptimal development under in vitro culture conditions.

  18. Gene expression patterns in granulosa cells and oocytes at various stages of follicle development as well as in in vitro grown oocyte-and-granulosa cell complexes

    PubMed Central

    MUNAKATA, Yasuhisa; KAWAHARA-MIKI, Ryoka; SHIRATSUKI, Shogo; TASAKI, Hidetaka; ITAMI, Nobuhiko; SHIRASUNA, Koumei; KUWAYAMA, Takehito; IWATA, Hisataka

    2016-01-01

    Follicle development is accompanied by proliferation of granulosa cells and increasing oocyte size. To obtain high-quality oocytes in vitro, it is important to understand the processes that occur in oocytes and granulosa cells during follicle development and the differences between in vivo and in vitro follicle development. In the present study, oocytes and granulosa cells were collected from early antral follicles (EAFs, 0.5–0.7 mm in diameter), small antral follicles (SAFs, 1–3 mm in diameter), large antral follicles (LAFs, 3–7 mm in diameter), and in vitro grown oocyte-and-granulosa cell complexes (OGCs), which were cultured for 14 days after collection from EAFs. Gene expression was analyzed comprehensively using the next-generation sequencing technology. We found top upstream regulators during the in vivo follicle development and compared them with those in in vitro developed OGCs. The comparison revealed that HIF1 is among the top regulators during both in vivo and in vitro development of OGCs. In addition, we found that HIF1-mediated upregulation of glycolysis in granulosa cells is important for the growth of OGCs, but the cellular metabolism differs between in vitro and in vivo grown OGCs. Furthermore, on the basis of comparison of upstream regulators between in vivo and in vitro development of OGCs, we believe that low expression levels of FLT1 (VEGFA receptor), SPP1, and PCSK6 can be considered causal factors of the suboptimal development under in vitro culture conditions. PMID:27108636

  19. Expression Analysis of Pluripotency Factors in the Undifferentiated Porcine Inner Cell Mass and Epiblast During In Vitro Culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Factors associated with pluripotency have been defined in mouse embryonic stem cells (mESC) and human embryonic stem cells (hESC). Some of these factors were expressed in-common, i.e., in both mESC and hESC, or in a species-specific manner. However, in the pig there is a much less complete underst...

  20. GLUT3 and PKM2 regulate OCT4 expression and support the hypoxic culture of human embryonic stem cells.

    PubMed

    Christensen, David R; Calder, Philip C; Houghton, Franchesca D

    2015-12-07

    Human embryonic stem cells (hESCs) have the capacity to differentiate into all cell types and thus have great potential for regenerative medicine. hESCs cultured at low oxygen tensions are more pluripotent and display an increased glycolytic rate but how this is regulated is unknown. This study therefore aimed to investigate the regulation of glucose metabolism in hESCs and whether this might impact OCT4 expression. In contrast to the glucose transporter GLUT1, GLUT3 was regulated by environmental oxygen and localised to hESC membranes. Silencing GLUT3 caused a reduction in glucose uptake and lactate production as well as OCT4 expression. GLUT3 and OCT4 expression were correlated suggesting that hESC self-renewal is regulated by the rate of glucose uptake. Surprisingly, PKM2, a rate limiting enzyme of glycolysis displayed a nuclear localisation in hESCs and silencing PKM2 did not alter glucose metabolism suggesting a role other than as a glycolytic enzyme. PKM2 expression was increased in hESCs cultured at 5% oxygen compared to 20% oxygen and silencing PKM2 reduced OCT4 expression highlighting a transcriptional role for PKM2 in hESCs. Together, these data demonstrate two separate mechanisms by which genes regulating glucose uptake and metabolism are involved in the hypoxic support of pluripotency in hESCs.

  1. Advanced APCVD-processes for high-temperature grown crystalline silicon thin film solar cells.

    PubMed

    Driessen, Marion; Merkel, Benjamin; Reber, Stefan

    2011-09-01

    Crystalline silicon thin film (cSiTF) solar cells based on the epitaxial wafer-equivalent (EpiWE) concept combine advantages of wafer-based and thin film silicon solar cells. In this paper two processes beyond the standard process sequence for cSiTF cell fabrication are described. The first provides an alternative to wet chemical saw damage removal by chemical vapor etching (CVE) with hydrogen chloride in-situ prior to epitaxial deposition. This application decreases the number of process and handling steps. Solar cells fabricated with different etching processes achieved efficiencies up to 14.7%. 1300 degrees C etching temperature led to better cell results than 1200 degrees C. The second investigated process aims for an improvement of cell efficiency by implementation of a reflecting interlayer between substrate and active solar cell. Some characteristics of epitaxial lateral overgrowth (ELO) of a patterned silicon dioxide film in a lab-type reactor constructed at Fraunhofer ISE are described and first solar cell results are presented.

  2. Changes in levels of cell wall constituents in wheat seedlings grown under continuous hypergravity conditions

    NASA Astrophysics Data System (ADS)

    Wakabayashi, K.; Soga, K.; Kamisaka, S.; Hoson, T.

    Effects of continuous hypergravity stimuli on the amounts and composition of cell wall constituents were investigated in wheat shoots. Hypergravity (300 g) treatment for three days after germination increased the net amount of cell wall polysaccharides such as hemicellulose and cellulose, but reduced the shoot elongation. As a result, the amount of cell wall polysaccharides per unit length of shoot increased under hypergravity. The hemicellulose fraction contained polysaccharides in the middle and low molecular mass range (5 kDa-1 MDa) and increased in response to hypergravity. Also, the amounts of arabinose (Ara) and xylose (Xyl), the major sugar components of the hemicellulose fraction, increased under hypergravity conditions. In addition to wall polysaccharides, hypergravity increased the amounts of cell wall-bound phenolic acids, such as ferulic acid (FA) and diferulic acid (DFA). Furthermore, the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was enhanced under hypergravity conditions. These results suggest that continuous hypergravity stimulates the synthesis of cell wall constituents, especially hemicellulosic arabinoxylans and cell wall-bound FA and DFA in wheat shoots. The increased PAL activity may promote the formation of FA and DFA. These changes in cell wall architecture may be involved in making rigid and tough cell walls under hypergravity conditions and thereby contribute to the ability of plant to sustain their structures against gravitational stimuli.

  3. Heteroepitaxial film silicon solar cell grown on Ni-W foils

    SciTech Connect

    Wee, Sung Hun; Cantoni, Claudia; Fanning, Thomas; Teplin, Charles; Bogorin, Daniela Florentina; Bornstein, Jon; Bowers, Karen; Schroeter,; Hasoon, Falah; Branz, Howard; Paranthaman, Mariappan Parans; Goyal, Amit

    2013-01-01

    Today, silicon-wafer-based technology dominates the photovoltaic (PV) industry because it enables high efficiency, is produced from abundant, non-toxic materials and is proven in the PV marketplace.[1] However, costs associated with the wafer itself limit ultimate cost reductions.[1,2] PV based on absorber layers of crystalline Si with only 2 to 10 m thickness are a promising route to reduce these costs, while maintaining efficiencies above 15%.[3-5] With the goal of fabricating low-cost film crystalline Si (c-Si), recent research has explored wafer peeling,[6,7] crystallization of amorphous silicon films on glass,[4,8-10] and seed and epitaxy approaches.[3,5,11] In this third approach, one initially forms a seed layer that establishes the grain size and crystalline order. The Si layer is then grown heteroepitaxially on the seed layer, so that it replicates the seed crystal structure. In all of these film c-Si approaches, the critical challenge is to grow c-Si with adequate material quality: specifically, the diffusion length (LD) must be at least three times the film thickness.[12] In polycrystalline Si films, grain boundaries (GBs) are recombination-active and significantly reduce LD. This adverse effects of GBs motivates research into growth of large grained c-Si [13,14] (for a low density of GBs) and biaxially-textured c-Si [11] (for low-angle GBs).

  4. On the association of human beta 2 microglobulin with cell culture-grown human cytomegalovirus (HCMV).

    PubMed

    Yamashita, Y; Shimokata, K; Nishiyama, Y

    1992-09-01

    We studied the production of human beta 2 microglobulin (beta 2m) in mock-infected or human cytomegalovirus (HCMV) infected human embryonic lung fibroblasts (HEL) and the association of human beta 2m with HCMV virions. Titration of beta 2m by two-step sandwich enzyme immunoassay revealed that HEL released considerable amounts of human beta 2m into the culture medium and that the production of beta 2m was significantly enhanced by HCMV infection. The concentration of human beta 2m in the culture medium of HCMV-infected HEL reached 500 to 600 ng/ml, which corresponded to 7- to 12-fold of levels found in healthy adult urine. Immunoprecipitation assays showed that HEL-grown HCMV bound a significant amount of endogenous beta 2m, but the viruses were efficiently neutralized by either human hyperimmune anti-HCMV globulin or anti-HCMV monoclonal antibody even when treated with a large amount of human beta 2m or with dialysed urine. Thus it seems unlikely that the binding of beta 2m by HCMV is involved in masking the viral antigenic site necessary for neutralization.

  5. Development of an in situ detachment protocol of Vero cells grown on Cytodex1 microcarriers under animal component-free conditions in stirred bioreactor.

    PubMed

    Rourou, Samia; Riahi, Nesrine; Majoul, Samy; Trabelsi, Khaled; Kallel, Héla

    2013-08-01

    Subcultivation of Vero cells grown in a proprietary animal component-free medium named IPT-AFM, on microcarriers, was studied. TrypLE Select, a non-animal-derived protease, was used as an alternative to trypsin for cell passaging. We first studied the effect of increasing concentrations of TrypLE Select toward cell growth and then studied the inactivation of the protease using either soybean trypsin inhibitor (STI) or the soy hydrolysate Hypep 1510, in six-well plates. Data showed that cell growth was impaired by residual level of TrypLE Select; STI was identified as an efficient agent to neutralize this effect. To restore cell growth and inactivate TrypLE Select, STI should be added to the medium at least at 0.2 g L(-1). Cells were also grown in spinner flask on 2 g L(-1) Cytodex1 in IPT-AFM. In these conditions, the cell detachment yield was equal to 78 ± 8 %. Furthermore, cells exhibited a typical growth profile when using the dislodged cells to seed a new culture. A cell detachment yield of 70 ± 19 % was also achieved when the cells were grown in a 2-L stirred bioreactor in IPT-AFM, on 3 g L(-1) Cytodex1. This protocol can be of great interest to scale-up the process of Vero cells cultivation in IPT-AFM on Cytodex1 from one stirred bioreactor culture to another.

  6. Transcriptome profiling in Arabidopsis inflorescence stems grown under hypergravity in terms of cell walls and plant hormones

    NASA Astrophysics Data System (ADS)

    Tamaoki, D.; Karahara, I.; Nishiuchi, T.; De Oliveira, S.; Schreiber, L.; Wakasugi, T.; Yamada, K.; Yamaguchi, K.; Kamisaka, S.

    2009-07-01

    Land plants rely on lignified secondary cell walls in supporting their body weight on the Earth. Although gravity influences the formation of the secondary cell walls, the regulatory mechanism of their formation by gravity is not yet understood. We carried out a comprehensive analysis of gene expression in inflorescence stems of Arabidopsis thaliana L. using microarray (22 K) to identify genes whose expression is modulated under hypergravity condition (300 g). Total RNA was isolated from the basal region of inflorescence stems of plants grown for 24 h at 300 g or 1 g. Microarray analysis showed that hypergravity up-regulated the expression of 403 genes to more than 2-fold. Hypergravity up-regulated the genes responsible for the biosynthesis or modification of cell wall components such as lignin, xyloglucan, pectin and structural proteins. In addition, hypergravity altered the expression of genes related to the biosynthesis of plant hormones such as auxin and ethylene and that of genes encoding hormone-responsive proteins. Our transcriptome profiling indicates that hypergravity influences the formation of secondary cell walls by modulating the pattern of gene expression, and that auxin and/or ethylene play an important role in signaling hypergravity stimulus.

  7. Effects of substrate conductivity on cell morphogenesis and proliferation using tailored, atomic layer deposition-grown ZnO thin films

    PubMed Central

    Choi, Won Jin; Jung, Jongjin; Lee, Sujin; Chung, Yoon Jang; Yang, Cheol-Soo; Lee, Young Kuk; Lee, You-Seop; Park, Joung Kyu; Ko, Hyuk Wan; Lee, Jeong-O

    2015-01-01

    We demonstrate that ZnO films grown by atomic layer deposition (ALD) can be employed as a substrate to explore the effects of electrical conductivity on cell adhesion, proliferation, and morphogenesis. ZnO substrates with precisely tunable electrical conductivity were fabricated on glass substrates using ALD deposition. The electrical conductivity of the film increased linearly with increasing duration of the ZnO deposition cycle (thickness), whereas other physical characteristics, such as surface energy and roughness, tended to saturate at a certain value. Differences in conductivity dramatically affected the behavior of SF295 glioblastoma cells grown on ZnO films, with high conductivity (thick) ZnO films causing growth arrest and producing SF295 cell morphologies distinct from those cultured on insulating substrates. Based on simple electrostatic calculations, we propose that cells grown on highly conductive substrates may strongly adhere to the substrate without focal-adhesion complex formation, owing to the enhanced electrostatic interaction between cells and the substrate. Thus, the inactivation of focal adhesions leads to cell proliferation arrest. Taken together, the work presented here confirms that substrates with high conductivity disturb the cell-substrate interaction, producing cascading effects on cellular morphogenesis and disrupting proliferation, and suggests that ALD-grown ZnO offers a single-variable method for uniquely tailoring conductivity. PMID:25897486

  8. Effects of substrate conductivity on cell morphogenesis and proliferation using tailored, atomic layer deposition-grown ZnO thin films.

    PubMed

    Choi, Won Jin; Jung, Jongjin; Lee, Sujin; Chung, Yoon Jang; Yang, Cheol-Soo; Lee, Young Kuk; Lee, You-Seop; Park, Joung Kyu; Ko, Hyuk Wan; Lee, Jeong-O

    2015-04-21

    We demonstrate that ZnO films grown by atomic layer deposition (ALD) can be employed as a substrate to explore the effects of electrical conductivity on cell adhesion, proliferation, and morphogenesis. ZnO substrates with precisely tunable electrical conductivity were fabricated on glass substrates using ALD deposition. The electrical conductivity of the film increased linearly with increasing duration of the ZnO deposition cycle (thickness), whereas other physical characteristics, such as surface energy and roughness, tended to saturate at a certain value. Differences in conductivity dramatically affected the behavior of SF295 glioblastoma cells grown on ZnO films, with high conductivity (thick) ZnO films causing growth arrest and producing SF295 cell morphologies distinct from those cultured on insulating substrates. Based on simple electrostatic calculations, we propose that cells grown on highly conductive substrates may strongly adhere to the substrate without focal-adhesion complex formation, owing to the enhanced electrostatic interaction between cells and the substrate. Thus, the inactivation of focal adhesions leads to cell proliferation arrest. Taken together, the work presented here confirms that substrates with high conductivity disturb the cell-substrate interaction, producing cascading effects on cellular morphogenesis and disrupting proliferation, and suggests that ALD-grown ZnO offers a single-variable method for uniquely tailoring conductivity.

  9. Treatment with a growth factor-protein mixture inhibits formation of mineralized nodules in osteogenic cell cultures grown on titanium.

    PubMed

    de Oliva, Marcos Andrade; Maximiano, William Marcatti Amarú; de Castro, Larissa Moreira Spínola; da Silva, Paulo Eliandro; Fernandes, Roger Rodrigo; Ciancaglini, Pietro; Beloti, Márcio Mateus; Nanci, Antonio; Rosa, Adalberto Luiz; de Oliveira, Paulo Tambasco

    2009-03-01

    Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. This study aimed to evaluate the effects of a well-defined PRP-like mixture containing platelet-derived growth factor-BB, transforming growth factor (TGF)-beta1, TGF-beta2, albumin, fibronectin, and thrombospondin [growth factors (GFs) + proteins] on the development of the osteogenic phenotype on titanium (Ti) in vitro. Human alveolar bone-derived osteoblastic cells were subcultured on Ti discs and exposed during the first 7 days to osteogenic medium supplemented with GFs + proteins and to osteogenic medium alone thereafter up to 14 days. Control cultures were exposed to only osteogenic medium. Dose-response experiments were carried out using rat primary calvarial cells exposed to GFs + proteins and 1:10 or 1:100 dilutions of the mixture. Treated human-derived cell cultures exhibited a significantly higher number of cycling cells at days 1 and 4 and of total cells at days 4 and 7, significantly reduced alkaline phosphatase (ALP) activity at days 4, 7, and 10, and no Alizarin red-stained areas (calcium deposits) at day 14, indicating an impairment in osteoblast differentiation. Although the 1:10 and 1:100 dilutions of the mixture restored the proliferative activity of rat-derived osteogenic cells to control levels and promoted a significant increase in ALP activity at day 10 compared with GFs + proteins, mineralized nodule formation was only observed with the 1:100 dilution ( approximately 50% of the control). These results showed that a PRP-like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblastic cell cultures grown on Ti.

  10. Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability

    PubMed Central

    Jacobs, Kurt; Zambelli, Filippo; Mertzanidou, Afroditi; Smolders, Ilse; Geens, Mieke; Nguyen, Ha Thi; Barbé, Lise; Sermon, Karen; Spits, Claudia

    2016-01-01

    Summary Human embryonic stem cells (hESC) show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term) impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem. PMID:26923824

  11. In vitro studies of cells grown on the superconductor PrO(x)FeAs.

    PubMed

    Yang, Shaoguang; Xie, Yuxuan; Yang, Wenrong; Zheng, Rongkun; Stevens, Frankie; Korkmaz, Emine; Weiss, Anthony S; Ringer, Simon P; Braet, Filip

    2009-06-01

    The recent discovery of arsenic-based high temperature superconductors has reignited interest in the study of superconductor: biological interfaces. However, the new superconductor materials involve the chemistry of arsenic and their toxicity remains unclear [Hand, E., 2008. Nature 452 (24), 922]. In this study the possible adverse effects of this new family of superconductors on cells have been examined. Cell culture studies in conjunction with microscopy and viability assays were employed to examine the influence of arsenic-based superconductor PrO(x)FeAs (x=0.75) material in vitro. Imaging data revealed that cells were well adhered and spread on the surface of the superconductor. Furthermore, cytotoxicity studies showed that cells were unaffected during the time-course of the experiments, providing support for the biocompatibility aspects of PrO(x)FeAs-based superconductor material.

  12. Investigation of InGaP/(In)AlGaAs/GaAs triple-junction top cells for smart stacked multijunction solar cells grown using molecular beam epitaxy

    NASA Astrophysics Data System (ADS)

    Sugaya, Takeyoshi; Mochizuki, Toru; Makita, Kikuo; Oshima, Ryuji; Matsubara, Koji; Okano, Yoshinobu; Niki, Shigeru

    2015-08-01

    We report high-quality InGaP/(In)AlGaAs/GaAs triple-junction solar cells fabricated using solid-source molecular beam epitaxy (MBE) for the first time. The triple-junction cells can be used as top cells for smart stacked multijunction solar cells. A growth temperature of 480 °C was found to be suitable for an (In)AlGaAs second cell to obtain high-quality tunnel junctions. The properties of AlGaAs solar cells were better than those of InAlGaAs solar cells when a second cell was grown at 480 °C. The high-quality InGaP/AlGaAs/GaAs solar cell had an impressive open-circuit voltage of 3.1 V. This result indicates that high-performance InGaP/AlGaAs/GaAs triple-junction solar cells can be fabricated using solid-source MBE.

  13. Changes in cell wall architecture of wheat coleoptiles grown under continuous hypergravity conditions

    NASA Astrophysics Data System (ADS)

    Wakabayashi, K.; Soga, K.; Kamisaka, S.; Hoson, T.

    Modifications of cell wall structure of wheat coleoptiles in response to continuous hypergravity (300 g) treatment were investigated. Length of coleoptiles exposed to hypergravity for 2-4 days from germination stage was 60-70% of that of 1 g control. The net amounts of cell wall polysaccharides, such as hemicellulose and cellulose, of hypergravity-treated coleoptiles increased as much as those of 1 g control coleoptiles during the incubation period. As a result, the levels of cell wall polysaccharides per unit length of coleoptile, which mean the thickness of cell walls, largely increased under hypergravity conditions. Particularly, the amounts of hemicellulosic polymers with middle molecular mass (0.2-1 MDa) largely increased from day 2 to 3 under hypergravity conditions. The major sugar components of the hemicellulose fraction are arabinose, xylose and glucose. The ratios of arabinose and xylose to glucose were higher in hypergravity-treated coleoptiles than in control coleoptiles. The fractionation of hemicellulosic polymers into the neutral and acidic polymers by the anion-exchange column showed that the levels of acidic polymers (mainly composed of arabinoxylans) in cell walls of hypergravity-treated coleoptiles were higher than those of control coleoptiles. In addition to wall polysaccharides, the amounts of cell wall-bound phenolics, such as ferulic acid and diferulic acid, substantially increased during the incubation period both in 1 g control and hypergravity-treated coleoptiles. Especially, the levels of diferulic acid which cross-links hemicellulosic polymers were higher in hypergravity-treated coleoptiles than in control coleoptiles during the incubation period. These results suggest that hypergravity stimuli from the germination stage bias the type of synthesized hemicellulosic polysaccharides, although they do not restrict the net synthesis of cell wall constituents in wheat coleoptiles. The stimulation of the synthesis of arabinoxylans and of the

  14. Amino Acid Pools and Metabolism During the Cell Division Cycle of Arginine-Grown Candida utilis

    PubMed Central

    Nurse, P.; Wiemken, A.

    1974-01-01

    Synchronous cultures obtained by isopycnic density gradient centrifugation are used to investigate amino acid metabolism during the cell division cycle of the food yeast Candida utilis. Isotopic labeling experiments demonstrate that the rates of uptake and catabolism of arginine, the sole source of nitrogen, double abruptly during the first half of the cycle, while the cells undergo bud expansion. This is accompanied by a doubling in rate of amino acid biosynthesis, and an accumulation of amino acids. The accumulation probably occurs within the storage pools of the vacuoles. Amino acids derived from protein degradation contribute little to this accumulation. For the remainder of the cell cycle, during cell separation and until the next bud initiation, the rates of uptake and catabolism of arginine and amino acid biosynthesis remain constant. Despite the abrupt doubling in the rate of formation of amino acid pools, their rate of utilization for macromolecular synthesis increases steadily throughout the cycle. The significance of this temporal organization of nitrogen source uptake and amino acid metabolism during the cell division cycle is discussed. Images PMID:4591945

  15. Generation of insulin-producing islet-like clusters from human embryonic stem cells.

    PubMed

    Jiang, Jianjie; Au, Melinda; Lu, Kuanghui; Eshpeter, Alana; Korbutt, Gregory; Fisk, Greg; Majumdar, Anish S

    2007-08-01

    Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to beta cells. Generation of glucose-responsive, insulin-producing beta cells from self-renewing, pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol, hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Ptf1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development, and the final population contained representatives of the ductal, exocrine, and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells, as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/mug of DNA was measured in the ILCs, representing levels higher than that of human fetal islets. In addition, the hESC-derived ILCs contained numerous secretory granules, as determined by electron microscopy, and secreted human C-peptide in a glucose-dependent manner. Disclosure of potential conflicts of interest is found at the end of this article.

  16. Photovoltaic characteristics of n(+)pp(+) InP solar cells grown by OMVPE

    NASA Technical Reports Server (NTRS)

    Tyagi, S.; Singh, K.; Bhimnathwala, H.; Ghandhi, S. K.; Borrego, J. M.

    1990-01-01

    The photovoltaic characteristics of n(+)/p/p(+) homojunction InP solar cells fabricated by organometallic vapor-phase epitaxy (OMVPE) are described. The cells are characterized by I-V, C-V and quantum efficiency measurements, and simulations are used to obtain various device and material parameters. The I-V characteristics show a high recombination rate in the depletion region; this is shown to be independent of the impurity used. It is shown that cadmium is easier to use as an acceptor for the p base and p(+) buffer and is therefore beneficial. The high quantum efficiency of 98 percent at long wavelengths measured in these cells indicates a very good collection efficiency in the base. The short-wavelength quantum efficiency is poor, indicating a high surface recombination.

  17. Optimization towards high density quantum dots for intermediate band solar cells grown by molecular beam epitaxy

    SciTech Connect

    Zhou, D.; Sharma, G.; Fimland, B. O.; Thomassen, S. F.; Reenaas, T. W.

    2010-02-08

    We report high density quantum dots (QDs) formation with optimized growth temperature and V/III ratio. At lower growth temperature, QD density is increased, due to smaller surface migration length of In adatoms. With higher V/III, the QD density is higher but it results in large clusters formation and decreases the QD uniformity. The QD solar cell was fabricated and examined. An extended spectral response in contrast to the GaAs reference cell was presented but the external quantum efficiency at energies higher than GaAs band gap is reduced, resulting from the degradation for the emitter above the strained QD layers.

  18. High efficiency GaAs-Ge tandem solar cells grown by MOCVD

    NASA Technical Reports Server (NTRS)

    Vernon, S. M.; Tobin, S. P.; Bajgar, C.; Haven, Victor E.; Geoffroy, L. M.; Lillington, D. R.; Hart, R. E., Jr.

    1989-01-01

    High conversion efficiency and low weight are obviously desirable for solar cells intended for space applications. One promising structure is GaAs on Ge. The advantages of using Ge wafers as substrates include the following: they offer high efficiency by forming a two-junction tandem cell; low weight combined with superior strength allows usage of thin (3 mil) wafers; and they are a good substrate for GaAs, being lattice matched, thermal expansion matched, and available as large-area wafers.

  19. Pluripotent states of human embryonic stem cells.

    PubMed

    Chen, Yifei; Lai, Dongmei

    2015-02-01

    Since human embryonic stem cells (hESCs) were first isolated and successfully cultured in vitro, the pluripotent potential of hESCs has been underestimated. The pluripotency of mouse embryonic stem cells (mESCs) can be categorized as naïve and primed, depending on their corresponding in vivo developing phases. mESC morphology differs at distinct pluripotent states, which differ in signaling dependence, gene expression, epigenetic features, and developmental potential. hESCs resemble mouse stem cells at primed pluripotency, and consequently are believed to correspond to a later developmental stage in vivo than mESCs. Nevertheless, recent studies indicate that a naïve state of pluripotency may exist in hESCs, and the pluripotency of hESCs also can be enhanced by genetic modification or optimized culture systems. These findings provide novel insight into the properties and differentiation potential of hESCs. Here, we review the recent advances in characterization of ESC states and investigate the mechanisms regulating hESC pluripotency. PMID:25393391

  20. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

    PubMed

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation. PMID:26378917

  1. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation. PMID:26378917

  2. Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood–derived hematopoietic progenitor cells

    PubMed Central

    Martin, Colin H.; Woll, Petter S.; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos

    2008-01-01

    Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34+ cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34+ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34+ cells also did not produce B cells in vitro. In contrast, CD34+ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor–induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs. PMID:18621931

  3. Heteroepitaxial Film Silicon Solar Cell Grown on Ni-W Foils

    SciTech Connect

    Wee, S. H.; Cantoni, C.; Fanning, T. R.; Teplin, C. W.; Bogorin, D. F.; Bornstein, J.; Bowers, K.; Schroeter, P.; Hasoon, F.; Branz, H. M.; Paranthaman, M. P.; Goyal, A.

    2012-03-01

    Heteroepitaxial semiconductor films on low-cost, flexible metal foil templates are a potential route to inexpensive, high-efficiency solar cells. Here, we report epitaxial growth of Si films on low-cost, flexible, biaxially-textured Ni-W substrates. A robust buffer architecture comprised of multiple epitaxial oxide layers has been developed to grow high quality, heteroepitaxial Si films without any undesired reaction between the Si film and the metal substrate and with a single biaxial texture. XRD analysis including {omega}-scans, {phi}-scans, and pole figures confirms that the buffers and silicon are all epitaxial, with excellent cube-on-cube epitaxy. A photo-conversion efficiency of 1.1% is demonstrated from a proof-of-concept heteroepitaxial film Si solar cell.

  4. Hybrid solar cells based on dc magnetron sputtered films of n-ITO on APMOVPE grown p-InP

    NASA Technical Reports Server (NTRS)

    Coutts, T. J.; Li, X.; Wanlass, M. W.; Emery, K. A.; Gessert, T. A.

    1988-01-01

    Hybrid indium-tin-oxide (ITO)/InP solar cells are discussed. The cells are constructed by dc magnetron sputter deposition of ITO onto high-quality InP films grown by atmospheric pressure metal-organic vapor-phase epitaxy (APMOVPE). A record efficiency of 18.9 percent, measured under standard Solar Energy Research Institute reporting conditions, has been obtained. The p-InP surface is shown to be type converted, principally by the ITO, but with the extent of conversion being modified by the nature of the sputtering gas. The deposition process, in itself, is not responsible for the type conversion. Dark currents have been suppressed by more than three orders of magnitude by the addition of hydrogen to the sputtering gas during deposition of a thin (5 nm) interface layer. Without this layer, and using only the more usual argon/oxygen mixture, the devices had poorer efficiencies and were unstable. A discussion of associated quantum efficiencies and capacitance/voltage measurements is also presented from which it is concluded that further improvements in efficiency will result from better control over the type-conversion process.

  5. Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

    PubMed Central

    Malpique, Rita; Brito, Catarina; Jensen, Janne; Bjorquist, Petter; Carrondo, Manuel J. T.; Alves, Paula M.

    2011-01-01

    The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications. PMID:21850261

  6. Performance of microbial electrolysis cells with bioanodes grown at different external resistances.

    PubMed

    Rago, Laura; Monpart, Nuria; Cortés, Pilar; Baeza, Juan A; Guisasola, Albert

    2016-01-01

    Bioelectrochemical systems need an anode with a high abundance of exoelectrogenic bacteria for an optimal performance. Among all possible operational parameters for an efficient enrichment, the role of external resistance in microbial fuel cell (MFC) has gained a lot of interest since it indirectly poises an anode potential, a key parameter for biofilm distribution and morphology. Thus, this work aims at investigating and discussing whether bioanodes selected at different external resistances under MFC operation present different responses under both MFC and microbial electrolysis cell (MEC) operation. A better MEC performance (i.e. shorter start-up time, higher current intensity and higher H2 production rate) was obtained with an anode from an MFC developed under low external resistance. Quantitative real-time polymerase chain reaction (qPCR) confirmed that a low external resistance provides an MFC anodic biofilm with the highest content of Geobacter because it allows higher current intensity, which is correlated to exoelectrogenic activity. High external resistances such as 1,000 Ω led to a slower start-up time under MEC operation.

  7. Performance of microbial electrolysis cells with bioanodes grown at different external resistances.

    PubMed

    Rago, Laura; Monpart, Nuria; Cortés, Pilar; Baeza, Juan A; Guisasola, Albert

    2016-01-01

    Bioelectrochemical systems need an anode with a high abundance of exoelectrogenic bacteria for an optimal performance. Among all possible operational parameters for an efficient enrichment, the role of external resistance in microbial fuel cell (MFC) has gained a lot of interest since it indirectly poises an anode potential, a key parameter for biofilm distribution and morphology. Thus, this work aims at investigating and discussing whether bioanodes selected at different external resistances under MFC operation present different responses under both MFC and microbial electrolysis cell (MEC) operation. A better MEC performance (i.e. shorter start-up time, higher current intensity and higher H2 production rate) was obtained with an anode from an MFC developed under low external resistance. Quantitative real-time polymerase chain reaction (qPCR) confirmed that a low external resistance provides an MFC anodic biofilm with the highest content of Geobacter because it allows higher current intensity, which is correlated to exoelectrogenic activity. High external resistances such as 1,000 Ω led to a slower start-up time under MEC operation. PMID:26942536

  8. GaAsPN-based PIN solar cells MBE-grown on GaP substrates: toward the III-V/Si tandem solar cell

    NASA Astrophysics Data System (ADS)

    Da Silva, M.; Almosni, S.; Cornet, C.; Létoublon, A.; Levallois, C.; Rale, P.; Lombez, L.; Guillemoles, J.-F.; Durand, O.

    2015-03-01

    GaAsPN semiconductors are promising material for the elaboration of high efficiencies tandem solar cells on silicon substrates. GaAsPN diluted nitride alloy is studied as the top junction material due to its perfect lattice matching with the Si substrate and its ideal bandgap energy allowing a perfect current matching with the Si bottom cell. We review our recent progress in materials development of the GaAsPN alloy and our recent studies of some of the different building blocks toward the elaboration of a PIN solar cell. A lattice matched (with a GaP(001) substrate, as a first step toward the elaboration on a Si substrate) 1μm-thick GaAsPN alloy has been grown by MBE. After a post-growth annealing step, this alloy displays a strong absorption around 1.8-1.9 eV, and efficient photoluminescence at room temperature suitable for the elaboration of the targeted solar cell top junction. Early stage GaAsPN PIN solar cells prototypes have been grown on GaP (001) substrates, with 2 different absorber thicknesses (1μm and 0.3μm). The external quantum efficiencies and the I-V curves show that carriers have been extracted from the GaAsPN alloy absorbers, with an open-circuit voltage of 1.18 V, while displaying low short circuit currents meaning that the GaAsPN structural properties needs a further optimization. A better carrier extraction has been observed with the absorber displaying the smallest thickness, which is coherent with a low carriers diffusion length in our GaAsPN compound. Considering all the pathways for improvement, the efficiency obtained under AM1.5G is however promising.

  9. Mosquito and mammalian cells grown on microcarriers for four-serotype dengue virus production: variations in virus titer, plaque morphology, and replication rate.

    PubMed

    Liu, Chia-Chyi; Wu, Suh-Chin

    2004-03-01

    Dengue (DEN) viruses consisting of four distinct serotypes cause diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Most of the dengue viruses can be effectively propagated in some mosquito and mammalian cell lines. In this study, we applied microcarrier cell culture technology to study two relevant aspects involving dengue virus, one on biotechnology of cell growth and virus production, and the other on virus biology concerning genetic variation of a virus population. We investigated the growth of C6/36 mosquito cells and Vero cells grown on Cytodex 1 microcarriers. High-titer DEN virus production can be achieved in C6/36 and Vero cells infected at low cell inoculation density, in the lag-phase cell stage, and at low multiplicity of infection (MOI). The maximum titers produced for DEN-1, DEN-3, and DEN-4 viruses were approximately 10- to 10,000-fold lower than for DEN-2 virus produced in C6/36 and Vero cells grown on microcarriers. The DEN-2 virus produced in C6/36 cells displayed far more extensive plaque heterogeneity than in Vero cells. Microcarrier C6/36 mosquito cell culture appeared to be the most effective system for four-serotype DEN virus production. Interestingly, some selected variants of DEN virus may outgrow in Vero cells when using a T-flask culture. These results may provide useful information for DEN vaccine development.

  10. Dye-sensitized solar cell employing zinc oxide aggregates grown in the presence of lithium

    DOEpatents

    Zhang, Qifeng; Cao, Guozhong

    2013-10-15

    Provided are a novel ZnO dye-sensitized solar cell and method of fabricating the same. In one embodiment, deliberately added lithium ions are used to mediate the growth of ZnO aggregates. The use of lithium provides ZnO aggregates that have advantageous microstructure, morphology, crystallinity, and operational characteristics. Employing lithium during aggregate synthesis results in a polydisperse collection of ZnO aggregates favorable for porosity and light scattering. The resulting nanocrystallites forming the aggregates have improved crystallinity and more favorable facets for dye molecule absorption. The lithium synthesis improves the surface stability of ZnO in acidic dyes. The procedures developed and disclosed herein also help ensure the formation of an aggregate film that has a high homogeneity of thickness, a high packing density, a high specific surface area, and good electrical contact between the film and the fluorine-doped tin oxide electrode and among the aggregate particles.

  11. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

    PubMed

    Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

    2015-09-01

    Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  12. Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

    NASA Technical Reports Server (NTRS)

    Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

    2011-01-01

    Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would

  13. A survey of parameters involved in the establishment of new lines of human embryonic stem cells.

    PubMed

    Fraga, Ana Maria; Souza de Araújo, Érica Sara; Stabellini, Raquel; Vergani, Naja; Pereira, Lygia V

    2011-11-01

    Since the derivation of the first human embryonic stem cell (hESC) lines by Thomson and coworkers in 1998, more than 1,200 different hESC lines have been established worldwide. Nevertheless, there is still a recognized interest in the establishment of new lines of hESC, particularly from HLA types and ethnic groups currently underrepresented among the available lines. The methodology of hESC derivation has evolved significantly since 1998, when human LIF (hLIF) was used for maintenance of pluripotency. However, there are a number of different strategies for the several steps involved in establishing a new line of hESC. Here we make a survey of the most relevant parameters used between 1998 and 2010 for the derivation of the 375 hESC lines deposited in two international stem cell registries, and able to form teratomas in immunocompromised mice. Although we identify some trends in the methodology for establishing hESC lines, our data reveal a much greater heterogeneity of strategies than what is used for derivation of murine ESC lines, indicating that optimum conditions have not been consolidated yet, and thus, hESC establishment is still an evolving field of research.

  14. Genetic processes in intergeneric cell hybrids Atropa + Nicotiana : 1. Genetic constitution of cells of different clonal origin grown in vitro.

    PubMed

    Gleba, Y Y; Momot, V P; Okolot, A N; Cherep, N N; Skarzhynskaya, M V; Kotov, V

    1983-06-01

    The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120-150 chromosomes including 50-70 of Atropa and 50-90 of Nicotiana. Other clones are polyploid and possess 200-250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.

  15. High-throughput tracking of pluripotent human embryonic stem cells with dual fluorescence resonance energy transfer molecular beacons.

    PubMed

    King, Frank W; Liszewski, Walter; Ritner, Carissa; Bernstein, Harold S

    2011-03-01

    Pluripotent human embryonic stem cells (hESCs) provide an unprecedented opportunity for the study of human tissue development, and the development of cell-based therapies for human disease. To realize these potential advances, however, methods for monitoring expression of intracellular proteins in live hESCs without altering cellular properties are needed. Molecular beacons are single-stranded oligonucleotides that have been employed to assay gene expression. To test their potential for high-throughput isolation of hESCs, we developed a dual fluorescence resonance energy transfer (FRET) molecular beacon system using fluorescence-activated cell sorting (FACS) with Oct4 as a target. We demonstrate that Oct4 can be detected by FRET using confocal microscopy, that this can be applied in a high-throughput manner to the identification and isolation of Oct4-expressing hESCs by FACS, that FRET-positive hESCs demonstrate pluripotency in culture and in vivo, and that hESCs transfected with molecular beacons demonstrate normal growth rates and oligonucleotide extinction over time. These studies demonstrate that FRET-based FACS using molecular beacons provides a useful tool for isolating Oct4-expressing pluripotent hESCs, and may also be adapted to selecting differentiating hESCs at specific developmental time points determined by transcription factor expression without functional or genomic alteration. As such, it provides an important new method for high-throughput isolation of hESC-derived tissue-specific precursors for analytic and therapeutic purposes.

  16. Sub-toxic concentrations of volatile organic compounds inhibit extracellular respiration of Escherichia coli cells grown in anodic bioelectrochemical systems.

    PubMed

    Santoro, Carlo; Mohidin, Abeed Fatima; Grasso, Letizia Lo; Seviour, Thomas; Palanisamy, Kannan; Hinks, Jamie; Lauro, Federico M; Marsili, Enrico

    2016-12-01

    Low-cost and rapid detection of volatile organic compounds (VOCs) is important for the control of water quality of used water and protection of downstream used water treatment processes. In this work, the effect of sub-toxic concentration of VOCs on the current output of Escherichia coli in bioelectrochemical systems (BES) is shown, in light of environmental sensing applications for sewage and used water networks. E. coli cells were grown on carbon felt electrodes in artificial used water, to increase sensitivity and decrease response time for detection. Extracellular electron transfer was promoted by the addition of a biocompatible redox mediator, 2-hydroxy-1,4-naphthoquinone (HNQ). Among the eight VOCs investigated, toluene is the most toxic to E. coli, with a detection limit of 50±2mgL(-1) and current output of 32±1nAmg(-1)L(-1). This work offers a straightforward route to enhance the detection of organic contaminants in used water for environmental applications. PMID:26947021

  17. Induction of tibial dyschondroplasia and suppression of cell-mediated immunity in chickens by Fusarium oxysporum grown on sterile corn.

    PubMed

    Chu, Q; Wu, W; Cook, M E; Smalley, E B

    1995-01-01

    An isolate of Fusarium oxysporum from corn associated with Kaschin-Beck disease in humans was tested for its ability to induce tibial dyschondroplasia (TD) and toxicity in chicks. Both leghorn and broiler chicks were fed diets in which corn was replaced with varied amounts (0% to 50%) of the F. oxysporum culture grown on sterile corn, or with known TD-inducing agents. F. oxysporum did not affect body weight in either type of chicks. In leghorn chicks, neither F. oxysporum nor the known TD-inducing agents (F. equiseti, 4%; tetramethylthiuram disulfide [Thiram], 35 ppm) caused TD. However, F. oxysporum at high levels (50%) and the two known TD-inducing agents reduced interdigital cutaneous response to phytohemagglutinin-P challenge. In addition, Thiram also reduced body-weight gain by more than 17%. In female broiler chicks (Cornish Rock), F. oxysporum not only decreased cell-mediated cutaneous response to phytohemagglutinin-P but also increased TD incidence; these same effects were observed with F. equiseti and Thiram. Histological examinations revealed similar pathological changes among dyschondroplastic lesions induced by F. oxysporum, F. equiseti, and Thiram. Results of this experiment indicate that the isolate of F. oxysporum from the region in which Kaschin-Beck disease is endemic can induce TD in broiler chicks and that it is immunosuppressive.

  18. Adenosine Signaling Mediates Osteogenic Differentiation of Human Embryonic Stem Cells on Mineralized Matrices

    PubMed Central

    Rao, Vikram; Shih, Yu-Ru V.; Kang, Heemin; Kabra, Harsha; Varghese, Shyni

    2015-01-01

    Human embryonic stem cells (hESCs) are attractive cell sources for tissue engineering and regenerative medicine due to their self-renewal and differentiation ability. Design of biomaterials with an intrinsic ability that promotes hESC differentiation to the targeted cell type boasts significant advantages for tissue regeneration. We have previously developed biomineralized calcium phosphate (CaP) matrices that inherently direct osteogenic differentiation of hESCs without the need of osteogenic-inducing chemicals or growth factors. Here, we show that CaP matrix-driven osteogenic differentiation of hESCs occurs through A2b adenosine receptor (A2bR). The inhibition of the receptor with an A2bR-specific antagonist attenuated mineralized matrix-mediated osteogenic differentiation of hESCs. In addition, when cultured on matrices in an environment deficient of CaP minerals, exogenous adenosine promoted osteogenic differentiation of hESCs, but was attenuated by the inhibition of A2bR. Such synthetic matrices that intrinsically support osteogenic commitment of hESCs are not only beneficial for bone tissue engineering but can also be used as a platform to study the effect of the physical and chemical cues to the extracellular milieu on stem cell commitment. Insights into the cell signaling during matrix-induced differentiation of stem cells will also help define the key processes and enable discovery of new targets that promote differentiation of pluripotent stem cells for bone tissue engineering. PMID:26618155

  19. Superparamagnetic iron oxide nanoparticles exert different cytotoxic effects on cells grown in monolayer cell culture versus as multicellular spheroids

    NASA Astrophysics Data System (ADS)

    Theumer, Anja; Gräfe, Christine; Bähring, Franziska; Bergemann, Christian; Hochhaus, Andreas; Clement, Joachim H.

    2015-04-01

    The aim of this study was to investigate the interaction of superparamagnetic iron oxide nanoparticles (SPION) with human blood-brain barrier-forming endothelial cells (HBMEC) in two-dimensional cell monolayers as well as in three-dimensional multicellular spheroids. The precise nanoparticle localisation and the influence of the NP on the cellular viability and the intracellular Akt signalling were studied in detail. Long-term effects of different polymer-coated nanoparticles (neutral fluidMAG-D, anionic fluidMAG-CMX and cationic fluidMAG-PEI) and the corresponding free polymers on cellular viability of HBMEC were investigated by real time cell analysis studies. Nanoparticles exert distinct effects on HBMEC depending on the nanoparticles' surface charge and concentration, duration of incubation and cellular context. The most severe effects were caused by PEI-coated nanoparticles. Concentrations above 25 μg/ml led to increased amounts of dead cells in monolayer culture as well as in multicellular spheroids. On the level of intracellular signalling, context-dependent differences were observed. Monolayer cultures responded on nanoparticle incubation with an increase in Akt phosphorylation whereas spheroids on the whole show a decreased Akt activity. This might be due to the differential penetration and distribution of PEI-coated nanoparticles.

  20. Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

    PubMed Central

    Geens, Mieke; Mateizel, Ileana; Sermon, Karen; De Rycke, Martine; Spits, Claudia; Cauffman, Greet; Devroey, Paul; Tournaye, Herman; Liebaers, Inge; Van de Velde, Hilde

    2009-01-01

    BACKGROUND Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent. PMID:19633307

  1. The influence of early embryo traits on human embryonic stem cell derivation efficiency.

    PubMed

    O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

    2011-05-01

    Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

  2. ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells.

    PubMed

    Adams, Bret R; Hawkins, Amy J; Povirk, Lawrence F; Valerie, Kristoffer

    2010-09-01

    We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes.

  3. Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.

    PubMed

    Finkbeiner, Stacy R; Freeman, Jennifer J; Wieck, Minna M; El-Nachef, Wael; Altheim, Christopher H; Tsai, Yu-Hwai; Huang, Sha; Dyal, Rachel; White, Eric S; Grikscheit, Tracy C; Teitelbaum, Daniel H; Spence, Jason R

    2015-10-12

    Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), called human intestinal organoids (HIOs), have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue.

  4. Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids

    PubMed Central

    Finkbeiner, Stacy R.; Freeman, Jennifer J.; Wieck, Minna M.; El-Nachef, Wael; Altheim, Christopher H.; Tsai, Yu-Hwai; Huang, Sha; Dyal, Rachel; White, Eric S.; Grikscheit, Tracy C.; Teitelbaum, Daniel H.; Spence, Jason R.

    2015-01-01

    ABSTRACT Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), called human intestinal organoids (HIOs), have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue. PMID:26459240

  5. Oxygen Partial Pressure Is a Rate-Limiting Parameter for Cell Proliferation in 3D Spheroids Grown in Physioxic Culture Condition

    PubMed Central

    Gomes, Aurélie; Guillaume, Ludivine; Grimes, David Robert; Fehrenbach, Jérôme; Lobjois, Valérie; Ducommun, Bernard

    2016-01-01

    The in situ oxygen partial pressure in normal and tumor tissues is in the range of a few percent. Therefore, when studying cell growth in 3D culture systems, it is essential to consider how the physiological oxygen concentration, rather than the one in the ambient air, influences the proliferation parameters. Here, we investigated the effect of reducing oxygen partial pressure from 21% to 5% on cell proliferation rate and regionalization in a 3D tumor spheroid model. We found that 5% oxygen concentration strongly inhibited spheroid growth, changed the proliferation gradient and reduced the 50% In Depth Proliferation index (IDP50), compared with culture at 21% oxygen. We then modeled the oxygen partial pressure profiles using the experimental data generated by culturing spheroids in physioxic and normoxic conditions. Although hypoxia occurred at similar depth in spheroids grown in the two conditions, oxygen partial pressure was a major rate-limiting factor with a critical effect on cell proliferation rate and regionalization only in spheroids grown in physioxic condition and not in spheroids grown at atmospheric normoxia. Our findings strengthen the need to consider conducting experiment in physioxic conditions (i.e., tissue normoxia) for proper understanding of cancer cell biology and the evaluation of anticancer drugs in 3D culture systems. PMID:27575790

  6. Action of calcitonin gene-related peptide upon bovine vascular endothelial and smooth muscle cells grown in isolation and co-culture.

    PubMed Central

    Crossman, D. C.; Dashwood, M. R.; Brain, S. D.; McEwan, J.; Pearson, J. D.

    1990-01-01

    1. Bovine aortic endothelial cells (BAE) and smooth muscle cells (BASM) were grown separately and in co-culture. 2. Calcitonin gene-related peptide (CGRP) caused dose-dependent activation of adenylate cyclase in each cell type when grown in isolation. The concentration of CGRP causing half-maximal activation in BAE and BASM was 200 nM and 310 nM, respectively. 3. In cells grown in co-culture exposure to bradykinin produced dose-dependent elevations in cyclic GMP content which were maximal 1 min after application of the agonist. 4. CGRP (1 nM-1 microM) did not stimulate a rise in cyclic GMP in co-cultures. 5. Displaceable CGRP binding was identified throughout the wall of the bovine aorta. 6. We conclude that CGRP receptors coupled to adenylate cyclase are present on BAE and BASM, but there is no coupling of these receptors to the release of any agent (such as endothelium-derived relaxing factor) that activates guanylate cyclase. Images Figure 1 Figure 2 PMID:2184911

  7. Oxygen Partial Pressure Is a Rate-Limiting Parameter for Cell Proliferation in 3D Spheroids Grown in Physioxic Culture Condition.

    PubMed

    Gomes, Aurélie; Guillaume, Ludivine; Grimes, David Robert; Fehrenbach, Jérôme; Lobjois, Valérie; Ducommun, Bernard

    2016-01-01

    The in situ oxygen partial pressure in normal and tumor tissues is in the range of a few percent. Therefore, when studying cell growth in 3D culture systems, it is essential to consider how the physiological oxygen concentration, rather than the one in the ambient air, influences the proliferation parameters. Here, we investigated the effect of reducing oxygen partial pressure from 21% to 5% on cell proliferation rate and regionalization in a 3D tumor spheroid model. We found that 5% oxygen concentration strongly inhibited spheroid growth, changed the proliferation gradient and reduced the 50% In Depth Proliferation index (IDP50), compared with culture at 21% oxygen. We then modeled the oxygen partial pressure profiles using the experimental data generated by culturing spheroids in physioxic and normoxic conditions. Although hypoxia occurred at similar depth in spheroids grown in the two conditions, oxygen partial pressure was a major rate-limiting factor with a critical effect on cell proliferation rate and regionalization only in spheroids grown in physioxic condition and not in spheroids grown at atmospheric normoxia. Our findings strengthen the need to consider conducting experiment in physioxic conditions (i.e., tissue normoxia) for proper understanding of cancer cell biology and the evaluation of anticancer drugs in 3D culture systems. PMID:27575790

  8. Identification of Proteins Related to Epigenetic Regulation in the Malignant Transformation of Aberrant Karyotypic Human Embryonic Stem Cells by Quantitative Proteomics

    PubMed Central

    Sun, Yi; Yang, Yixuan; Zeng, Sicong; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

    2014-01-01

    Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs. PMID:24465727

  9. Comparison of the antiproliferative activity of crude ethanol extracts of nine salvia species grown in Jordan against breast cancer cell line models

    PubMed Central

    Abu-Dahab, Rana; Afifi, Fatma; Kasabri, Violet; Majdalawi, Lara; Naffa, Randa

    2012-01-01

    Background: The antiproliferative activity of Salvia species grown in Jordan has not been fully evaluated yet. The aim of this work was to study the antiproliferative activity of crude ethanol extracts from nine Salvia species grown in Jordan against a panel of breast cancer cell lines. Material and Methods: Cytotoxic activity was evaluated in human tumor models of breast cancer; MCF-7, T47D, ZR-75-1, and BT 474 by the sulforhodamine B assay. In addition, the extracts were evaluated using a non-transformed cell line (Vero) and normal fibroblast cells in order to demonstrate their selectivity and safety. Results: From the nice ethanol extracts under investigation, those of S. dominica and S. fruticosa showed an inhibitory concentration of 50% of cells (IC50) in concentrations less than 30μg/mL against the four cell lines under investigation. S. syriaca and S. hormium showed an IC50 below 30μg/ml for two out of the four cell lines. S. fruticosa, S. hormium and S. syriaca showed selectivity in their antiproliferative activity against estrogen receptor positive cell lines with minimal toxicity against normal human periodontal fibroblasts. Phytochemical screening using thin layer chromatography indicated the presence of terpenoids, flavonoids and coumarins in all examined extracts. Conclusion: Three of the plant extracts under investigation exhibited antiproliferative activity against breast cancer cells and were shown to be safe and selective. These could be considered as a potential source for novel anticancer therapy. PMID:24082637

  10. Polymer-based protein engineering grown ferrocene-containing redox polymers improve current generation in an enzymatic biofuel cell.

    PubMed

    Campbell, Alan S; Murata, Hironobu; Carmali, Sheiliza; Matyjaszewski, Krzysztof; Islam, Mohammad F; Russell, Alan J

    2016-12-15

    Enzymatic biofuel cells (EBFCs) are capable of generating electricity from physiologically present fuels making them promising power sources for the future of implantable devices. The potential application of such systems is limited, however, by inefficient current generation. Polymer-based protein engineering (PBPE) offers a unique method to tailor enzyme function through tunable modification of the enzyme surface with functional polymers. In this study, we report on the modification of glucose oxidase (GOX) with ferrocene-containing redox polymers to increase current generation efficiency in an enzyme-modified anode. Poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide) (pFcAc) was grown from covalently attached, water-soluble initiator molecules on the surface of GOX in a "grafting-from" approach using atom transfer radical polymerization (ATRP). The covalently-coupled ferrocene-containing polymers on the enzyme surface promoted the effective "wiring" of the GOX active site to an external electrode. The resulting GOX-pFcAc conjugates generated over an order of magnitude increase in current generation efficiency and a 4-fold increase in maximum EBFC power density (≈1.7µWcm(-2)) with similar open circuit voltage (0.27V) compared to native GOX when physically adsorbed onto paddle-shaped electrodes made up of electrospun polyacrylonitrile fibers coated with gold nanoparticles and multi-wall carbon nanotubes. The formation of electroactive enzyme-redox polymer conjugates using PBPE represents a powerful new tool for the improvement of mediated enzyme-based bioelectronics without the need for free redox mediators or anode/cathode compartmentalization.

  11. Polymer-based protein engineering grown ferrocene-containing redox polymers improve current generation in an enzymatic biofuel cell.

    PubMed

    Campbell, Alan S; Murata, Hironobu; Carmali, Sheiliza; Matyjaszewski, Krzysztof; Islam, Mohammad F; Russell, Alan J

    2016-12-15

    Enzymatic biofuel cells (EBFCs) are capable of generating electricity from physiologically present fuels making them promising power sources for the future of implantable devices. The potential application of such systems is limited, however, by inefficient current generation. Polymer-based protein engineering (PBPE) offers a unique method to tailor enzyme function through tunable modification of the enzyme surface with functional polymers. In this study, we report on the modification of glucose oxidase (GOX) with ferrocene-containing redox polymers to increase current generation efficiency in an enzyme-modified anode. Poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide) (pFcAc) was grown from covalently attached, water-soluble initiator molecules on the surface of GOX in a "grafting-from" approach using atom transfer radical polymerization (ATRP). The covalently-coupled ferrocene-containing polymers on the enzyme surface promoted the effective "wiring" of the GOX active site to an external electrode. The resulting GOX-pFcAc conjugates generated over an order of magnitude increase in current generation efficiency and a 4-fold increase in maximum EBFC power density (≈1.7µWcm(-2)) with similar open circuit voltage (0.27V) compared to native GOX when physically adsorbed onto paddle-shaped electrodes made up of electrospun polyacrylonitrile fibers coated with gold nanoparticles and multi-wall carbon nanotubes. The formation of electroactive enzyme-redox polymer conjugates using PBPE represents a powerful new tool for the improvement of mediated enzyme-based bioelectronics without the need for free redox mediators or anode/cathode compartmentalization. PMID:27424262

  12. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    SciTech Connect

    Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva; Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs; Apati, Agota

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  13. Efficient large-scale generation of functional hepatocytes from mouse embryonic stem cells grown in a rotating bioreactor with exogenous growth factors and hormones

    PubMed Central

    2013-01-01

    Introduction Embryonic stem (ES) cells are considered a potentially advantageous source of hepatocytes for both transplantation and the development of bioartificial livers. However, the efficient large-scale generation of functional hepatocytes from ES cells remains a major challenge, especially for those methods compatible with clinical applications. Methods In this study, we investigated whether a large number of functional hepatocytes can be differentiated from mouse ES (mES) cells using a simulated microgravity bioreactor. mES cells were cultured in a rotating bioreactor in the presence of exogenous growth factors and hormones to form embryoid bodies (EBs), which then differentiated into hepatocytes. Results During the rotating culture, most of the EB-derived cells gradually showed the histologic characteristics of normal hepatocytes. More specifically, the expression of hepatic genes and proteins was detected at a higher level in the differentiated cells from the bioreactor culture than in cells from a static culture. On further growing, the EBs on tissue-culture plates, most of the EB-derived cells were found to display the morphologic features of hepatocytes, as well as albumin synthesis. In addition, the EB-derived cells grown in the rotating bioreactor exhibited higher levels of liver-specific functions, such as glycogen storage, cytochrome P450 activity, low-density lipoprotein, and indocyanine green uptake, than did differentiated cells grown in static culture. When the EB-derived cells from day-14 EBs and the cells’ culture supernatant were injected into nude mice, the transplanted cells were engrafted into the recipient livers. Conclusions Large quantities of high-quality hepatocytes can be generated from mES cells in a rotating bioreactor via EB formation. This system may be useful in the large-scale generation of hepatocytes for both cell transplantation and the development of bioartificial livers. PMID:24294908

  14. Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies.

    PubMed

    Moon, Sung-Hwan; Ju, Jongil; Park, Soon-Jung; Bae, Daekyeong; Chung, Hyung-Min; Lee, Sang-Hoon

    2014-07-01

    Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell-cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.

  15. No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

    PubMed Central

    Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi

    2010-01-01

    Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828

  16. Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system.

    PubMed

    Aflatoonian, Behrouz; Ruban, Ludmila; Shamsuddin, Shamsul; Baker, Duncan; Andrews, Peter; Moore, Harry

    2010-04-01

    The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 microl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs. PMID:20224972

  17. Changes of ribulose bisphosphate carboxylase/oxygenase content, ribulose bisphosphate concentration, and photosynthetic activity during adaptation of high-CO/sub 2/ grown cells to low-CO/sub 2/ conditions in Chlorella pyrenoidosa

    SciTech Connect

    Yokota, A.; Canvin, D.T.

    1986-02-01

    Changes of some photosynthetic properties of high-CO/sub 2/ grown cells of Chlorella pyrenoidosa during adaptation to low-CO/sub 2/ conditions have been investigated. The K/sub m/ value of photosynthesis of the high-CO/sub 2/ grown cells for dissolved inorganic carbon was 3.3 millimolar and decreased to 25 to 30 micromolar within 4 hours after transferring to air. In the presence of saturating CO/sub 2/ concentrations the photosynthetic activity of the high-CO/sub 2/ grown cells was 1.5 times as high as that of the low-CO/sub 2/ grown cells. There was a significant rise of the photosynthetic activity during adaptation of the high-CO/sub 2/ grown cells to air, followed by a steady decrease. The activity of ribulose 1,5-bisphosphate carboxylase/oxygenase in both the high and low-CO/sub 2/ grown cells was close to the photosynthetic activity of the cells. The concentration of ribulose 1,5-bisphosphate (RuBP) was higher in the low-CO/sub 2/ adapting and low-CO/sub 2/ grown celsl than in the high-CO/sub 2/ grown cells regardless of the photosynthetic rate. This seems to be due to an increased RuBP regeneration activity during adaptation followed by maintenance of the new higher concentration. The RuBP level always exceeded the concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase RuBP binding sites in both the high- and low-CO/sub 2/ grown cells at any dissolved inorganic carbon concentration.

  18. X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells.

    PubMed

    Silva, Susana S; Rowntree, Rebecca K; Mekhoubad, Shila; Lee, Jeannie T

    2008-03-25

    With the potential to give rise to all somatic cell types, human embryonic stem cells (hESC) have generated enormous interest as agents of cell replacement therapy. One potential limitation is their safety in vivo. Although several studies have focused on concerns over genomic stability ex vivo, few have analyzed epigenetic stability. Here, we use tools of the epigenetic phenomenon, X-chromosome inactivation (XCI), to investigate their epigenetic properties. Among 11 distinct hESC lines, we find a high degree of variability. We show that, like mouse ESC, hESC in principle have the capacity to recapitulate XCI when induced to differentiate in culture (class I lines). However, this capacity is seen in few hESC isolates. Many hESC lines have already undergone XCI (class II and III). Unexpectedly, there is a tendency to lose XIST RNA expression during culture (class III). Despite losing H3-K27 trimethylation, the inactive X of class III lines remains transcriptionally suppressed, as indicated by Cot-1 RNA exclusion. We conclude that hESC lines are subject to dynamic epigenetic reprogramming ex vivo. Given that XCI and cell differentiation are tightly linked, we consider implications for hESC pluripotency and differentiation potential.

  19. X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells

    PubMed Central

    Silva, Susana S.; Rowntree, Rebecca K.; Mekhoubad, Shila; Lee, Jeannie T.

    2008-01-01

    With the potential to give rise to all somatic cell types, human embryonic stem cells (hESC) have generated enormous interest as agents of cell replacement therapy. One potential limitation is their safety in vivo. Although several studies have focused on concerns over genomic stability ex vivo, few have analyzed epigenetic stability. Here, we use tools of the epigenetic phenomenon, X-chromosome inactivation (XCI), to investigate their epigenetic properties. Among 11 distinct hESC lines, we find a high degree of variability. We show that, like mouse ESC, hESC in principle have the capacity to recapitulate XCI when induced to differentiate in culture (class I lines). However, this capacity is seen in few hESC isolates. Many hESC lines have already undergone XCI (class II and III). Unexpectedly, there is a tendency to lose XIST RNA expression during culture (class III). Despite losing H3-K27 trimethylation, the inactive X of class III lines remains transcriptionally suppressed, as indicated by Cot-1 RNA exclusion. We conclude that hESC lines are subject to dynamic epigenetic reprogramming ex vivo. Given that XCI and cell differentiation are tightly linked, we consider implications for hESC pluripotency and differentiation potential. PMID:18339803

  20. Patents on Technologies of Human Tissue and Organ Regeneration from Pluripotent Human Embryonic Stem Cells

    PubMed Central

    Parsons, Xuejun H; Teng, Yang D; Moore, Dennis A; Snyder, Evan Y

    2011-01-01

    Human embryonic stem cells (hESCs) are genetically stable with unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of a large supply of disease-targeted human somatic cells that are restricted to the lineage in need of repair. There is a large healthcare need to develop hESC-based therapeutic solutions to provide optimal regeneration and reconstruction treatment options for the damaged or lost tissue or organ that have been lacking. In spite of controversy surrounding the ownership of hESCs, the number of patent applications related to hESCs is growing rapidly. This review gives an overview of different patent applications on technologies of derivation, maintenance, differentiation, and manipulation of hESCs for therapies. Many of the published patent applications have been based on previously established methods in the animal systems and multi-lineage inclination of pluripotent cells through spontaneous germ-layer differentiation. Innovative human stem cell technologies that are safe and effective for human tissue and organ regeneration in the clinical setting remain to be developed. Our overall view on the current patent situation of hESC technologies suggests a trend towards hESC patent filings on novel therapeutic strategies of direct control and modulation of hESC pluripotent fate, particularly in a 3-dimensional context, when deriving clinically-relevant lineages for regenerative therapies. PMID:23355961

  1. Responses of human embryonic stem cells and their differentiated progeny to ionizing radiation

    SciTech Connect

    Zou, Ying; Zhang, Ningzhe; Ellerby, Lisa M.; Davalos, Albert R.; Zeng, Xianmin; Campisi, Judith; Desprez, Pierre-Yves

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer hESCs and their progeny, NSCs and neurons, were exposed to ionizing radiation. Black-Right-Pointing-Pointer Upon irradiation, most hESCs died within 5-7 h. Black-Right-Pointing-Pointer Surviving NSCs underwent senescence and displayed features of astrocytes. Black-Right-Pointing-Pointer Surviving NSCs did not display the secretory phenotype expressed by pure astrocytes. Black-Right-Pointing-Pointer This study is to better understand the stress-responses of hESCs and their progeny. -- Abstract: Human embryonic stem cells (hESCs) hold promise for the treatment of many human pathologies. For example, hESCs and the neuronal stem cells (NSCs) and neurons derived from them have significant potential as transplantation therapies for a variety of neurodegenerative diseases. Two concerns about the use of hESCs and their differentiated derivatives are their ability to function and their ability to resist neoplastic transformation in response to stresses that inevitably arise during their preparation for transplantation. To begin to understand how these cells handle genotoxic stress, we examined the responses of hESCs and derived NSCs and neurons to ionizing radiation (IR). Undifferentiated hESCs were extremely sensitive to IR, with nearly all the cells undergoing cell death within 5-7 h. NSCs and neurons were substantially more resistant to IR, with neurons showing the most resistant. Of interest, NSCs that survived IR underwent cellular senescence and acquired astrocytic characteristics. Unlike IR-treated astrocytes, however, the NSC-derived astrocytic cells that survived IR did not display the typical pro-inflammatory, pro-carcinogenic senescence-associated secretory phenotype. These findings suggest distinct genotoxic stress-responses of hESCs and derived NSC and neuronal populations, and suggest that damaged NSCs, while failing to function, may not cause local inflammation.

  2. Direct Conversion of Pluripotent Human Embryonic Stem Cells Under Defined Culture Conditions into Human Neuronal or Cardiomyocyte Cell Therapy Derivatives.

    PubMed

    Parsons, Xuejun H

    2016-01-01

    Developing novel strategies for well-controlled efficiently directing pluripotent human embryonic stem cells (hESCs) exclusively and uniformly towards clinically relevant cell types in a lineage-specific manner is not only crucial for unveiling the molecular and cellular cues that direct human embryogenesis but also vital to harnessing the power of hESC biology for tissue engineering and cell-based therapies. Conventional hESC differentiation methods require uncontrollable simultaneous multi-lineage differentiation of pluripotent cells, which yield embryoid bodies (EB) or aggregates consisting of a mixed population of cell types of three embryonic germ layers, among which only a very small fraction of cells display targeted differentiation, impractical for commercial and clinical applications. Here, a protocol for lineage-specific differentiation of hESCs, maintained under defined culture systems, direct from the pluripotent stage using small-molecule induction exclusively and uniformly to a neural or a cardiac lineage is described. Lineage-specific differentiation of pluripotent hESCs by small-molecule induction enables well-controlled highly efficient direct conversion of nonfunctional pluripotent hESCs into a large supply of high-purity functional human neuronal or cardiomyocyte cell therapy derivatives for commercial and therapeutic uses.

  3. Alginate Microfiber System for Expansion and Direct Differentiation of Human Embryonic Stem Cells.

    PubMed

    Leong, Meng Fatt; Lu, Hong Fang; Lim, Tze Chiun; Narayanan, Karthikeyan; Gao, Shujun; Wang, Luna Yue; Toh, Rebecca P K; Funke, Henrik; Abdul Samad, Muhammad Hamizan; Wan, Andrew C A; Ying, Jackie Y

    2016-09-01

    Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications, there is a need to develop scalable cell culture platforms to propagate hESCs. In this study, we encapsulated hESCs in calcium alginate microfibers as single cells, for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (>10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM), which did not affect hESCs viability, was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical, as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line, HUES7, for up to five passages while maintaining pluripotency. Immunohistochemistry, polymerase chain reaction, and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells, possessed normal karyotype, and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively, our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions. PMID:27484909

  4. Dopaminergic Differentiation of Human Embryonic Stem Cells on PA6-Derived Adipocytes.

    PubMed

    Guloglu, M Oktar; Larsen, Anna

    2016-01-01

    Human embryonic stem cells (hESCs) are a promising source for cell replacement therapies. Parkinson's disease is one of the candidate diseases for the cell replacement therapy since the motor manifestations of the disease are associated with the loss of dopaminergic neurons in the substantia nigra pars compacta. Stromal cell-derived inducing activity (SDIA) is the most commonly used method for the dopaminergic differentiation of hESCs. This chapter describes a simple, reliable, and scalable dopaminergic induction method of hESCs using PA6-derived adipocytes. Coculturing hESCs with PA6-derived adipocytes markedly reduces the variable outcomes among experiments. Moreover, the colony differentiation step of this method can also be used for the dopaminergic induction of mouse embryonic stem cells and NTERA2 cells as well.

  5. Magnesium doping of efficient GaAs and Ga(0.75)In(0.25)As solar cells grown by metalorganic chemical vapor deposition

    NASA Technical Reports Server (NTRS)

    Lewis, C. R.; Ford, C. W.; Werthen, J. G.

    1984-01-01

    Magnesium has been substituted for zinc in GaAs and Ga(0.75)In(0.25)As solar cells grown by metalorganic chemical vapor deposition (MOCVD). Bis(cyclopentadienyl)magnesium (Cp2Mg) is used as the MOCVD transport agent for Mg. Full retention of excellent material quality and efficient cell performance results. The substitution of Mg for Zn would enhance the abruptness and reproducibility of doping profiles, and facilitate high temperature processing and operation, due to the much lower diffusion coefficient of Mg, relative to Zn, in these materials.

  6. A multiple p-n junction structure obtained from as-grown Czochralski silicon crystals by heat treatment - Application to solar cells

    NASA Technical Reports Server (NTRS)

    Chi, J. Y.; Gatos, H. C.; Mao, B. Y.

    1980-01-01

    Multiple p-n junctions have been prepared in as-grown Czochralski p-type silicon through overcompensation near the oxygen periodic concentration maxima by oxygen thermal donors generated during heat treatment at 450 C. Application of the multiple p-n-junction configuration to photovoltaic energy conversion has been investigated. A new solar-cell structure based on multiple p-n-junctions was developed. Theoretical analysis showed that a significant increase in collection efficiency over the conventional solar cells can be achieved.

  7. The Enhancement of Human Embryonic Stem Cell Osteogenic Differentiation with Nano-Fibrous Scaffolding

    PubMed Central

    Smith, Laura A.; Liu, Xiaohua; Hu, Jiang; Ma, Peter X.

    2010-01-01

    Human embryonic stem cells (hESC) hold great promise as a cell source for tissue engineering since they possess the ability to differentiate into any cell type within the body. However, much work must still be done to control the differentiation of the hESC to the desired lineage. In this study, we examined the effects of the nano-fibrous (NF) architecture in both two dimensional (2-D) poly(L-lactic acid) (PLLA) thin matrices and 3-D PLLA scaffolds in vitro to assess their affect on the osteogenic differentiation of hESC in vitro compared to more traditional solid films and solid-walled (SW) scaffolds. In 2-D culture, hESC on NF thin matrices were found to express collagen type 1, Runx2, and osteocalcin mRNA of higher levels than the hESC on the solid films after 1 week of culture and increased mineralization was observed on the NF matrices compared to the solid films after 3 weeks of culture. After 6 weeks of 3-D culture, the hESC on the NF scaffolds expressed signifcantly more osteocalcin mRNA compared to these on the SW scaffolds. The data indicates that the NF architecture enhances the osteogenic differentiation of the hESC compared to more traditional scaffolding architecture. PMID:20430439

  8. VGLL4 is a Novel Regulator of Survival in Human Embryonic Stem Cells

    PubMed Central

    Tajonar, Adriana; Maehr, René; Hu, Guang; Sneddon, Julie B.; Rivera-Feliciano, José; Cohen, Dena E.; Elledge, Stephen J.; Melton, Douglas A.

    2014-01-01

    Human embryonic stem cells (hESCs) are maintained in a self-renewing state by an interconnected network of mechanisms that sustain pluripotency, promote proliferation and survival, and prevent differentiation. We sought to find novel genes that could contribute to one or more of these processes by using a gain-of-function screen of a large collection of human open reading frames. We identified Vestigial-like 4 (VGLL4), a co-transcriptional regulator with no previously described function in hESCs, as a positive regulator of survival in hESCs. Specifically, VGLL4 overexpression in hESCs significantly decreases cell death in response to dissociation stress. Additionally, VGLL4 overexpression enhances hESC colony formation from single cells. These effects may be attributable, in part, to a decreased activity of initiator and effector caspases observed in the context of VGLL4 overexpression. Additionally, we show an interaction between VGLL4 and the Rho/Rock pathway, previously implicated in hESC survival. This study introduces a novel gain-of-function approach for studying hESC maintenance and presents VGLL4 as a previously undescribed regulator of this process. PMID:23765749

  9. Power-Laws and the Use of Pluripotent Stem Cell Lines

    PubMed Central

    Lenz, Michael; Kobold, Sabine; MacArthur, Ben D.; Schuppert, Andreas; Löser, Peter; Müller, Franz-Josef

    2013-01-01

    It is widely accepted that the (now reversed) Bush administration’s decision to restrict federal funding for human embryonic stem cell (hESC) research to a few “eligible” hESC lines is responsible for the sustained preferential use of a small subset of hESC lines (principally the H1 and H9 lines) in basic and preclinical research. Yet, international hESC usage patterns, in both permissive and restrictive political environments, do not correlate with a specific type of stem cell policy. Here we conducted a descriptive analysis of hESC line usage and compared the ability of policy-driven processes and collaborative processes inherent to biomedical research to recapitulate global hESC usage patterns. We find that current global hESC usage can be modelled as a cumulative advantage process, independent of restrictive or permissive policy influence, suggesting a primarily innovation-driven (rather than policy-driven) mechanism underlying human pluripotent stem cell usage in preclinical research. PMID:23300961

  10. ZnO nanowires array grown on Ga-doped ZnO single crystal for dye-sensitized solar cells

    PubMed Central

    Hu, Qichang; Li, Yafeng; Huang, Feng; Zhang, Zhaojun; Ding, Kai; Wei, Mingdeng; Lin, Zhang

    2015-01-01

    High quality ZnO nanowires arrays were homoepitaxial grown on Ga-doped ZnO single crystal (GZOSC), which have the advantages of high conductivity, high carrier mobility and high thermal stability. When it was employed as a photoanode in the DSSCs, the cell exhibited a 1.44% power-conversion efficiency under the illumination of one sun (AM 1.5G). The performance is superior to our ZnO nanowires/FTO based DSSCs under the same condition. This enhanced performance is mainly attributed to the perfect interface between the ZnO nanowires and the GZOSC substrate that contributes to lower carrier scattering and recombination rates compared with that grown on traditional FTO substrate. PMID:26099568

  11. Epithelial cell adhesion molecule regulation is associated with the maintenance of the undifferentiated phenotype of human embryonic stem cells.

    PubMed

    Lu, Tung-Ying; Lu, Ruei-Min; Liao, Mei-Ying; Yu, John; Chung, Chu-Hung; Kao, Cheng-Fu; Wu, Han-Chung

    2010-03-19

    Human embryonic stem cells (hESCs) are unique pluripotent cells capable of self-renewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumor-initiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (mAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation analysis to elucidate the direct regulation by EpCAM of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2, and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs. PMID:20064925

  12. Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

    PubMed

    Tsai, Steven C; Chang, David F; Hong, Chang-Mu; Xia, Ping; Senadheera, Dinithi; Trump, Lisa; Mishra, Suparna; Lutzko, Carolyn

    2014-06-15

    Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

  13. A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs.

    PubMed

    Choi, Jiho; Lee, Soohyun; Mallard, William; Clement, Kendell; Tagliazucchi, Guidantonio Malagoli; Lim, Hotae; Choi, In Young; Ferrari, Francesco; Tsankov, Alexander M; Pop, Ramona; Lee, Gabsang; Rinn, John L; Meissner, Alexander; Park, Peter J; Hochedlinger, Konrad

    2015-11-01

    The equivalence of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) remains controversial. Here we use genetically matched hESC and hiPSC lines to assess the contribution of cellular origin (hESC vs. hiPSC), the Sendai virus (SeV) reprogramming method and genetic background to transcriptional and DNA methylation patterns while controlling for cell line clonality and sex. We find that transcriptional and epigenetic variation originating from genetic background dominates over variation due to cellular origin or SeV infection. Moreover, the 49 differentially expressed genes we detect between genetically matched hESCs and hiPSCs neither predict functional outcome nor distinguish an independently derived, larger set of unmatched hESC and hiPSC lines. We conclude that hESCs and hiPSCs are molecularly and functionally equivalent and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional and epigenetic comparisons of pluripotent cell lines, explaining some of the previously observed differences between genetically unmatched hESCs and hiPSCs. PMID:26501951

  14. GaAs Solar Cells Grown by Hydride Vapor-Phase Epitaxy and the Development of GaInP Cladding Layers

    SciTech Connect

    Simon, John; Schulte, Kevin L.; Young, David L.; Haegel, Nancy M.; Ptak, Aaron J.

    2016-01-01

    The high cost of high-efficiency III-V photovoltaic devices currently limits them to niche markets. Hydride vapor-phase epitaxy (HVPE) growth of III-V materials recently reemerged as a low-cost, high-throughput alternative to conventional metal- organic vapor-phase epitaxy (MOVPE) growth of high-efficiency solar cells. Previously, we demonstrated unpassivated HVPEgrown GaAs p-n junctions with good quantum efficiency and high open-circuit voltage (Voc). In this work, we demonstrate the growth of GaInPby HVPE for use as a high-quality surface passivation layer to GaAs solar cells. Solar cells grown with GaInP window layers show significantly improved quantum efficiency compared with unpassivated cells, increasing the short-circuit current (JSC) of these low-cost devices. These results show the potential of low-cost HVPE for the growth of high-quality III-V devices.

  15. Borate cross-linked/total rhamnogalacturonan II ratio in cell walls for the biochemical diagnosis of boron deficiency in hydroponically grown pumpkin.

    PubMed

    Matsunaga, Toshiro; Ishii, Tadashi

    2006-08-01

    Boron (B) is an essential micronutrient for vascular plants. The function of B has been demonstrated to cross-link monomeric rhamnogalacturonan II (mRG-II) to form dimeric RG-II-borate (dRG-II-B), and thus to stabilize plant cell walls. The dRG-II-B to total RG-II ratio in the cell walls of pumpkin hydroponically grown under various low-B conditions was analyzed to evaluate its applicability to the diagnosis of plant B deficiency. The dRG-II-B ratio in cell walls ranged between approximately 0.9 in B-sufficient tissues and approximately 0.15 in severe B-deficient tissues, reflecting the B nutritional status of tissues. This result indicates that the degree of B shortage in plant tissues is very likely to be diagnosed by the dRG-II-B ratio in cell walls.

  16. Power recovery of radiation damaged MOCVD grown indium phosphide on silicon solar cells through argon-ion laser annealing. Master`s thesis

    SciTech Connect

    Boyer, L.L.

    1996-06-01

    This thesis reports the results of a laser annealing technique used to remove defect sites from radiation damaged indium phosphide on silicon MOCVD grown solar cells. This involves the illumination of damaged solar cells with a continuous wave laser to produce a large forward-biased current. The InP/Si cells were irradiated with 1 MeV electrons to a given fluence, and tested for degradation. Light from an argon laser was used to illuminate four cells with an irradiance of 2.5 W/sq cm, producing a current density 3 to 5 times larger than AMO conditions. Cells were annealed at 19 deg C with the laser and at 25 deg C under AMO conditions. Annealing under laser illumination of n/p-type cells resulted in recovery of 48%. P/n type cells lost 4 to 12% of the assumed degradaton. Annealing under AMO conditions resulted in power recovery of 70% in n/p type cells. P/n-type cells recovered approximately 16% of lost power. Results indicate that significant power recovery results from the annealing of defects within n/p type InP/Si solar cells.

  17. Synthetic niches for differentiation of human embryonic stem cells bypassing embryoid body formation.

    PubMed

    Liu, Yarong; Fox, Victoria; Lei, Yuning; Hu, Biliang; Joo, Kye-Il; Wang, Pin

    2014-07-01

    The unique self-renewal and pluripotency features of human embryonic stem cells (hESCs) offer the potential for unlimited development of novel cell therapies. Currently, hESCs are cultured and differentiated using methods, such as monolayer culture and embryoid body (EB) formation. As such, achieving efficient differentiation into higher order structures remains a challenge, as well as maintaining cell viability during differentiation into homogeneous cell populations. Here, we describe the application of highly porous polymer scaffolds as synthetic stem cell niches. Bypassing the EB formation step, these scaffolds are capable of three-dimensional culture of undifferentiated hESCs and subsequent directed differentiation into three primary germ layers. H9 hESCs were successfully maintained and proliferated in biodegradable polymer scaffolds based on poly (lactic-co-glycolic acid) (PLGA). The results showed that cells within PLGA scaffolds retained characteristics of undifferentiated pluripotent stem cells. Moreover, the scaffolds allowed differentiation towards the lineage of interest by the addition of growth factors to the culture system. The in vivo transplantation study revealed that the scaffolds could provide a microenvironment that enabled hESCs to interact with their surroundings, thereby promoting cell differentiation. Therefore, this approach, which provides a unique culture/differentiation system for hESCs, will find its utility in various stem cell-based tissue-engineering applications.

  18. Epigenetics changes caused by the fusion of human embryonic stem cell and ovarian cancer cells.

    PubMed

    He, Ke; Qu, Hu; Xu, Li-Nan; Gao, Jun; Cheng, Fu-Yi; Xiang, Peng; Zhou, Can-Quan

    2016-10-01

    To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer.

  19. Epigenetics changes caused by the fusion of human embryonic stem cell and ovarian cancer cells.

    PubMed

    He, Ke; Qu, Hu; Xu, Li-Nan; Gao, Jun; Cheng, Fu-Yi; Xiang, Peng; Zhou, Can-Quan

    2016-10-01

    To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer. PMID:27377320

  20. Epigenetics changes caused by the fusion of human embryonic stem cell and ovarian cancer cells

    PubMed Central

    He, Ke; Qu, Hu; Xu, Li-Nan; Gao, Jun; Cheng, Fu-Yi; Xiang, Peng; Zhou, Can-Quan

    2016-01-01

    To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer. PMID:27377320

  1. Synergistic effect of dual interfacial modifications with room-temperature-grown epitaxial ZnO and adsorbed indoline dye for ZnO nanorod array/P3HT hybrid solar cell.

    PubMed

    Chen, Dian-Wei; Wang, Ting-Chung; Liao, Wen-Pin; Wu, Jih-Jen

    2013-09-11

    ZnO nanorod (NR)/poly(3-hexylthiophene) (P3HT) hybrid solar cells with interfacial modifications are investigated in this work. The ZnO NR arrays are modified with room-temperature (RT)-grown epitaxial ZnO shells or/and D149 dye molecules prior to the P3HT infiltration. A synergistic effect of the dual modifications on the efficiency of the ZnO NR/P3HT solar cell is observed. The open-circuit voltage and fill factor are considerable improved through the RT-grown ZnO and D149 modifications in sequence on the ZnO NR array, which brings about a 2-fold enhancement of the efficiency of the ZnO NR/P3HT solar cell. We suggested that the more suitable surface of RT-grown ZnO for D149 adsorption, the chemical compatibility of D149 and P3HT, and the elevated conduction band edge of the RT-grown ZnO/D149-modified ZnO NR array construct the superior interfacial morphology and energetics in the RT-grown ZnO/D149-modified ZnO NR/P3HT hybrid solar cell, resulting in the synergistic effect on the cell efficiency. An efficiency of 1.16% is obtained in the RT-grown ZnO/D149-modified ZnO NR/P3HT solar cell. PMID:23937447

  2. Human umbilical cord Wharton's jelly stem cells undergo enhanced chondrogenic differentiation when grown on nanofibrous scaffolds and in a sequential two-stage culture medium environment.

    PubMed

    Fong, Chui-Yee; Subramanian, Arjunan; Gauthaman, Kalamegam; Venugopal, Jayarama; Biswas, Arijit; Ramakrishna, Seeram; Bongso, Ariff

    2012-03-01

    The current treatments used for osteoarthritis from cartilage damage have their disadvantages of donor site morbidity, complicated surgical interventions and risks of infection and graft rejection. Recent advances in tissue engineering have offered much promise in cartilage repair but the best cell source and in vitro system have not as yet been optimised. Human bone marrow mesenchymal stem cells (hBMSCs) have thus far been the cell of choice. However, we derived a unique stem cell from the human umbilical cord Wharton's jelly (hWJSC) that has properties superior to hBMSCs in terms of ready availability, prolonged stemness characteristics in vitro, high proliferation rates, wide multipotency, non-tumorigenicity and tolerance in allogeneic transplantation. We observed enhanced cell attachment, cell proliferation and chondrogenesis of hWJSCs over hBMSCs when grown on PCL/Collagen nanoscaffolds in the presence of a two-stage sequential complex/chondrogenic medium for 21 days. Improvement of these three parameters were confirmed via inverted optics, field emission scanning electron microscopy (FESEM), MTT assay, pellet diameters, Alcian blue histology and staining, glycosaminglycans (GAG) and hyaluronic acid production and expression of key chondrogenic genes (SOX9, Collagen type II, COMP, FMOD) using immunohistochemistry and real-time polymerase chain reaction (qRT-PCR). In separate experiments we demonstrated that the 16 ng/ml of basic fibroblast growth factor (bFGF) present in the complex medium may have contributed to driving chondrogenesis. We conclude that hWJSCs are an attractive stem cell source for inducing chondrogenesis in vitro when grown on nanoscaffolds and exposed sequentially first to complex medium and then followed by chondrogenic medium.

  3. Biological impact of human embryonic stem cells.

    PubMed

    Martín, Miguel; Menéndez, Pablo

    2012-01-01

    Research on human embryonic stem cells (hESCs) and induced pluripotent (iPS) stem cells is currently a field of great potential in biomedicine. These cells represent a highly valuable tool for developmental biology studies, disease models, and drug screening and toxicity. The ultimate goal of hESCs and iPS cell research is the treatment of diseases or disorders for which there is currently no treatment or existing therapies are only partially effective. Despite the disproportionate short-term hopes generated, which are putting too much pressure on scientists, the international scientific community is making rapid progress in understanding hESCs and iPS cells. Nonetheless, great efforts have to be made to provide an answer to still quite basic questions concerning their biology. Moreover, translation to clinical applications in cell replacement therapy requires prior solution to ethical barriers. The recent development of iPS cells has provided a strong alternative to overcome ethical issues concerning hESCs. However, an in-depth characterization of their genetic and epigenetic features, as well as their differentiation potential still remains to be undertaken. This chapter will describe, precisely, what the critical issues are, where scientific and ethical barriers stand, and how we are to overcome them. Only then, we shall finally discover whether hESCs and iPS cells will allow building reproducible disease models, and whether they really are a safe tool, with great potential for regenerative medicine.

  4. Alginate Encapsulation of Human Embryonic Stem Cells to Enhance Directed Differentiation to Pancreatic Islet-Like Cells

    PubMed Central

    Richardson, Thomas; Kumta, Prashant N.

    2014-01-01

    The pluripotent property of human embryonic stem cells (hESCs) makes them attractive for treatment of degenerative diseases such as diabetes. We have developed a stage-wise directed differentiation protocol to produce alginate-encapsulated islet-like cells derived from hESCs, which can be directly implanted for diabetes therapy. The advantage of alginate encapsulation lies in its capability to immunoisolate, along with the added possibility of scalable culture. We have evaluated the possibility of encapsulating hESCs at different stages of differentiation. Encapsulation of predifferentiated cells resulted in insufficient cellular yield and differentiation. On the other hand, encapsulation of undifferentiated hESCs followed by differentiation induction upon encapsulation resulted in the highest viability and differentiation. More striking was that alginate encapsulation resulted in a much stronger differentiation compared to parallel two-dimensional cultures, resulting in 20-fold increase in c-peptide protein synthesis. To elucidate the mechanism contributing to encapsulation-mediated enhancement in hESC maturation, investigation of the signaling pathways revealed interesting insight. While the phospho-protein levels of all the tested signaling molecules were lower under encapsulation, the ratio of pSMAD/pAKT was significantly higher, indicating a more efficient signal transduction under encapsulation. These results clearly demonstrate that alginate encapsulation of hESCs and differentiation to islet-cell types provides a potentially translatable treatment option for type 1 diabetes. PMID:24881778

  5. Grown-in defects and defects produced by 1-Me electron irradiated in Al0.3Ga0.7As P-N junction solar cells

    NASA Technical Reports Server (NTRS)

    Li, S. S.; Teng, K. W.; Schoenfeld, D. W.; Rahilly, W. P.

    1982-01-01

    Studies of grown-in defects and defects produced by the one-MeV electron irradiation in Al sub 0.3 Ga sub 0.7As p-n junction solar cells fabricated by liquid phase epitaxial (LPE) technique were made for the unirradiated and one-MeV electron irradiated samples, using DLTS and C-V methods. Defect and recombination parameters such as energy level, defect density, carrier capture cross sections and lifetimes were determined for various growth, annealing, and irradiation conditions.

  6. Cellular response to the deposition of diesel exhaust particle aerosols onto human lung cells grown at the air-liquid interface by inertial impaction.

    PubMed

    Cooney, Daniel J; Hickey, Anthony J

    2011-12-01

    The pathogenesis of disease resulting from exposure to diesel exhaust particles (DEP) is often studied using cultured lung cells. Frequently, researchers expose cells to DEP by spiking a suspension of particles in liquid onto the apical surface. This is not representative of in vivo exposure, where aerosols are deposited onto cell surfaces at the air-liquid interface (ALI). Inertial impaction provides an opportunity to deliver high doses of particles with aerodynamic diameters>∼1 μm to the surface of cells in seconds in a reproducible and predictable manner. A custom device was constructed to deposit DEP aerosols onto the surface of Calu-3 and A549 cells grown at the ALI. The pro-inflammatory and toxic cellular response to exposure to the deposited DEP aerosols was measured and compared to the response of cells exposed to DEP as suspensions. Calu-3 cells showed evidence of an oxidative stress response for both exposure types, while there was strong evidence to suggest that the method of aerosol delivery was harmful to the A549 cells.

  7. Human Lung Cancer Cells Grown in an Ex Vivo 3D Lung Model Produce Matrix Metalloproteinases Not Produced in 2D Culture

    PubMed Central

    Mishra, Dhruva K.; Sakamoto, Jason H.; Thrall, Michael J.; Baird, Brandi N.; Blackmon, Shanda H.; Ferrari, Mauro; Kurie, Jonathan M.; Kim, Min P.

    2012-01-01

    We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture. PMID:23028922

  8. Human Embryonic Stem Cells: A Model for the Study of Neural Development and Neurological Diseases

    PubMed Central

    Prajumwongs, Piya; Weeranantanapan, Oratai; Jaroonwitchawan, Thiranut; Noisa, Parinya

    2016-01-01

    Although the mechanism of neurogenesis has been well documented in other organisms, there might be fundamental differences between human and those species referring to species-specific context. Based on principles learned from other systems, it is found that the signaling pathways required for neural induction and specification of human embryonic stem cells (hESCs) recapitulated those in the early embryo development in vivo at certain degree. This underscores the usefulness of hESCs in understanding early human neural development and reinforces the need to integrate the principles of developmental biology and hESC biology for an efficient neural differentiation. PMID:27239201

  9. Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

    PubMed

    Hertsenberg, Andrew J; Funderburgh, James L

    2016-01-01

    Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

  10. Progress in human embryonic stem cell research in the United States between 2001 and 2010.

    PubMed

    Vakili, Keyvan; McGahan, Anita M; Rezaie, Rahim; Mitchell, Will; Daar, Abdallah S

    2015-01-01

    On August 9th, 2001, the federal government of the United States announced a policy restricting federal funds available for research on human embryonic stem cell (hESCs) out of concern for the "vast ethical mine fields" associated with the creation of embryos for research purposes. Until the policy was repealed on March 9th, 2009, no U.S. federal funds were available for research on hESCs extracted after August 9, 2001, and only limited federal funds were available for research on a subset of hESC lines that had previously been extracted. This paper analyzes how the 2001 U.S. federal funding restrictions influenced the quantity and geography of peer-reviewed journal publications on hESC. The primary finding is that the 2001 policy did not have a significant aggregate effect on hESC research in the U.S. After a brief lag in early 2000s, U.S. hESC research maintained pace with other areas of stem cell and genetic research. The policy had several other consequences. First, it was tied to increased hESC research funding within the U.S. at the state level, leading to concentration of related activities in a relatively small number of states. Second, it stimulated increased collaborative research between US-based scientists and those in countries with flexible policies toward hESC research (including Canada, the U.K., Israel, China, Spain, and South Korea). Third, it encouraged independent hESC research in countries without restrictions.

  11. Progress in Human Embryonic Stem Cell Research in the United States between 2001 and 2010

    PubMed Central

    Vakili, Keyvan; McGahan, Anita M.; Rezaie, Rahim; Mitchell, Will; Daar, Abdallah S.

    2015-01-01

    On August 9th, 2001, the federal government of the United States announced a policy restricting federal funds available for research on human embryonic stem cell (hESCs) out of concern for the “vast ethical mine fields” associated with the creation of embryos for research purposes. Until the policy was repealed on March 9th, 2009, no U.S. federal funds were available for research on hESCs extracted after August 9, 2001, and only limited federal funds were available for research on a subset of hESC lines that had previously been extracted. This paper analyzes how the 2001 U.S. federal funding restrictions influenced the quantity and geography of peer-reviewed journal publications on hESC. The primary finding is that the 2001 policy did not have a significant aggregate effect on hESC research in the U.S. After a brief lag in early 2000s, U.S. hESC research maintained pace with other areas of stem cell and genetic research. The policy had several other consequences. First, it was tied to increased hESC research funding within the U.S. at the state level, leading to concentration of related activities in a relatively small number of states. Second, it stimulated increased collaborative research between US-based scientists and those in countries with flexible policies toward hESC research (including Canada, the U.K., Israel, China, Spain, and South Korea). Third, it encouraged independent hESC research in countries without restrictions. PMID:25812114

  12. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells.

    PubMed

    Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T; Henningsen, Jeanette; Kratchmarova, Irina; Kassem, Moustapha; Blagoev, Blagoy

    2009-05-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.

  13. Self-renewal of human embryonic stem cells on defined synthetic electrospun nanofibers.

    PubMed

    Kumar, Deepak; Dale, Tina P; Yang, Ying; Forsyth, Nicholas R

    2015-12-01

    Human embryonic stem cells (hESCs) are conventionally expanded and maintained in vitro on biological substrates. Synthetic electrospun polymer nanofibers have the potential to act as non-biological substrates in the culture of hESCs. Three synthetic, FDA approved polymers: poly-ɛ-caprolactone (PCL), poly-L-lactic acid (PLLA) and poly lactic-co-glycolic acid (PLGA) were electrospun as nanofibers (random or aligned conformations) on glass coverslips and their supportive role in hESC culture examined. Clonogenicity experiments demonstrated that nanofibrous scaffolds (PCL aligned and random, PLLA aligned and PLGA aligned) supported hESC adhesion and expansion. A significantly greater number of colonies were observed on PCL-aligned nanofibrous scaffolds in comparison to PLLA-aligned and PLGA-aligned substrates (p < 0.05). hESC colonies were significantly larger on PCL aligned nanofibrous substrates when compared to other polymer substrates (p < 0.05-0.001), where fiber diameter played a pivotal role in support of hESC clonogenicity (on PCL). Retention of pluripotentiality was confirmed by expression of Alkaline phosphatase, OCT-3/4 and Nanog expression on PCL scaffolds and the expression of transcripts representative of mesoderm (ACTC1), ectoderm (SOX1) and endoderm (AFP) during subsequent spontaneous in vitro differentiation. These results demonstrate the potential of nanofibers as xeno-free scaffolds supportive of hESC adhesion, self-renewal and differentiation in in vitro culture conditions. PMID:26610861

  14. Comparison of single junction AlGaInP and GaInP solar cells grown by molecular beam epitaxy

    SciTech Connect

    Masuda, T; Tomasulo, S; Lang, JR; Lee, ML

    2015-03-07

    We have investigated similar to 2.0 eV (AlxGa1-x)(0.51)In0.49P and similar to 1.9 eV Ga0.51In0.49P single junction solar cells grown on both on-axis and misoriented GaAs substrates by molecular beam epitaxy (MBE). Although lattice-matched (AlxGa1-x)(0.51)In0.49P solar cells are highly attractive for space and concentrator photovoltaics, there have been few reports on the MBE growth of such cells. In this work, we demonstrate open circuit voltages (V-oc) ranging from 1.29 to 1.30 V for Ga0.51In0.49P cells, and 1.35-1.37 V for (AlxGa1-x)(0.51)In0.49P cells. Growth on misoriented substrates enabled the bandgap-voltage offset (W-oc = E-g/q - V-oc) of Ga0.51In0.49P cells to decrease from similar to 575 mV to similar to 565 mV, while that of (AlxGa1-x)(0.51)In0.49P cells remained nearly constant at 620 mV. The constant Woc as a function of substrate offcut for (AlxGa1-x)(0.51)In0.49P implies greater losses from non-radiative recombination compared with the Ga0.51In0.49P devices. In addition to larger Woc values, the (AlxGa1-x)(0.51)In0.49P cells exhibited significantly lower internal quantum efficiency (IQE) values than Ga0.51In0.49P cells due to recombination at the emitter/window layer interface. A thin emitter design is experimentally shown to be highly effective in improving IQE, particularly at short wavelengths. Our work shows that with further optimization of both cell structure and growth conditions, MBE-grown (AlxGa1-x)(0.51)In0.49P will be a promising wide-bandgap candidate material for high-efficiency, lattice-matched multi-junction solar cells. (C) 2015 AIP Publishing LLC.

  15. Automated, scalable culture of human embryonic stem cells in feeder-free conditions.

    PubMed

    Thomas, Rob J; Anderson, David; Chandra, Amit; Smith, Nigel M; Young, Lorraine E; Williams, David; Denning, Chris

    2009-04-15

    Large-scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However, current hESC culture strategies are labor-intensive and employ highly variable processes, presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines, HUES7, and NOTT1, with trypsin in feeder-free conditions, is compatible with complete automation on the CompacT SelecT, a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained, as evidenced by consistent proliferation during serial passage, expression of stem cell markers (OCT4, NANOG, TRA1-81, and SSEA-4), stable karyotype, and multi-germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell-use in clinical, scientific, and industrial applications.

  16. Significant changes in cell and chloroplast development in young wheat leaves (Triticum aestivum cv Hereward) grown in elevated CO{sub 2}

    SciTech Connect

    Robertson, E.J.; Leech, R.M.

    1995-01-01

    Cell and chloroplast development were characterized in young Triticum aestivum cv Hereward leaves grown at ambient (350 {mu}L L{sup {minus}1}) or at elevated (650 {mu}L L{sup {minus}1}) CO{sub 2}. In elevated CO{sub 2}, cell and chloroplast expansion was accelerated by 10 and 25%, respectively, in the first leaf of 7-d-old wheat plants without disruption to the leaf developmental pattern. Elevated CO{sub 2} did not affect the number of chloroplasts in relation to mesophyll cell size or the linear relationship between chloroplast number or size and mesophyll cell size. No major changes in leaf anatomy or in chloroplast ultrastructure were detected as a result of growth in elevated CO{sub 2}, but there was a marked reduction in starch accumulation. In leaf sections fluorescently tagged antisera were used to visualize and quantitate the amount of cytochrome f, the {alpha}- and {beta}-subunits of the coupling factor 1 in ATP synthase, D1 protein of the photosystem II reaction center, the 33-kD protein of the extrinsic oxygen-evolving complex, subunit II of photosystem I, and ribulose-1,5-biphosphate carboxylase/oxygenase. A significant finding was that in 10 to 20% of the mesophyll cells grown in elevated CO{sub 2} the 33-kD protein of the extrinsic oxygen-evolving complex of photosystem II and cytochrome f were deficient by 75%, but the other proteins accumulated normally. 29 refs., 6 figs., 2 tabs.

  17. Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control.

    PubMed

    Serra, Margarida; Brito, Catarina; Sousa, Marcos F Q; Jensen, Janne; Tostões, Rui; Clemente, João; Strehl, Raimund; Hyllner, Johan; Carrondo, Manuel J T; Alves, Paula M

    2010-08-01

    The successful transfer of human embryonic stem cell (hESC) technology and cellular products into clinical and industrial applications needs to address issues of automation, standardization and the generation of relevant cell numbers of high quality. In this study, we combined microcarrier technology and controlled stirred tank bioreactors, to develop an efficient and scalable system for expansion of pluripotent hESCs. We demonstrate the importance of controlling pO(2) at 30% air saturation to improve hESCs growth. This concentration allowed for a higher energetic cell metabolism, increased growth rate and maximum cell concentration in contrast to 5% pO(2) where a shift to anaerobic metabolism was observed, decreasing cell expansion 3-fold. Importantly, the incorporation of an automated perfusion system in the bioreactor enhanced culture performance and allowed the continuous addition of small molecules assuring higher cell concentrations for a longer time period. The expanded hESCs retained their undifferentiated phenotype and pluripotency. Our results show, for the first time, that the use of controlled bioreactors is critical to ensure the production of high quality hESCs. When compared to the standard colony culture, our strategy improves the final yield of hESCs by 12-fold, providing a potential bioprocess to be transferred to clinical and industrial applications.

  18. Rare cell proteomic reactor applied to stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics study of human embryonic stem cell differentiation.

    PubMed

    Tian, Ruijun; Wang, Shuai; Elisma, Fred; Li, Li; Zhou, Hu; Wang, Lisheng; Figeys, Daniel

    2011-02-01

    The molecular basis governing the differentiation of human embryonic stem cells (hESCs) remains largely unknown. Systems-level analysis by proteomics provides a unique approach to tackle this question. However, the requirement of a large number of cells for proteomics analysis (i.e. 10(6)-10(7) cells) makes this assay challenging, especially for the study of rare events during hESCs lineage specification. Here, a fully integrated proteomics sample processing and analysis platform, termed rare cell proteomic reactor (RCPR), was developed for large scale quantitative proteomics analysis of hESCs with ∼50,000 cells. hESCs were completely extracted by a defined lysis buffer, and all of the proteomics sample processing procedures, including protein preconcentration, reduction, alkylation, and digestion, were integrated into one single capillary column with a strong cation exchange monolith matrix. Furthermore, on-line two-dimensional LC-MS/MS analysis was performed directly using RCPR as the first dimension strong cation exchange column. 2,281 unique proteins were identified on this system using only 50,000 hESCs. For stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative study, a ready-to-use and chemically defined medium and an in situ differentiation procedure were developed for complete SILAC labeling of hESCs with well characterized self-renewal and differentiation properties. Mesoderm-enriched differentiation was studied by RCPR using 50,000 hESCs, and 1,086 proteins were quantified with a minimum of two peptides per protein. Of these, 56 proteins exhibited significant changes during mesoderm-enriched differentiation, and eight proteins were demonstrated for the first time to be overexpressed during early mesoderm development. This work provides a new platform for the study of rare cells and in particular for further elucidating proteins that govern the mesoderm lineage specification of human pluripotent stem cells.

  19. High-efficiency solar cells on edge-defined film-fed grown (18.2%) and string ribbon (17.8%) silicon by rapid thermal processing

    NASA Astrophysics Data System (ADS)

    Rohatgi, A.; Kim, D. S.; Nakayashiki, K.; Yelundur, V.; Rounsaville, B.

    2004-01-01

    Solar cell efficiencies of 18.2 and 17.8% were achieved on edge-defined film-fed grown and string ribbon multicrystalline silicon, respectively. Improved understanding and hydrogenation of defects in ribbon materials contributed to the significant increase in bulk lifetime from 1-5 μs to as high as 90-100 μs during cell processing. It was found that SiNx-induced defect hydrogenation in these ribbon materials takes place within one second at 740-750 °C. The bulk lifetime decreases at annealing temperatures above 750 °C or annealing times above one second due to the enhanced dissociation of the hydrogenated defects coupled with the decrease in hydrogen supply from the SiNx film deposited by plasma enhanced chemical vapor deposition.

  20. Properties of Retinal Precursor Cells Grown on Vertically Aligned Multiwalled Carbon Nanotubes Generated for the Modification of Retinal Implant-Embedded Microelectrode Arrays

    PubMed Central

    Johnen, Sandra; Meißner, Frank; Krug, Mario; Baltz, Thomas; Endler, Ingolf; Mokwa, Wilfried; Walter, Peter

    2016-01-01

    Background. To analyze the biocompatibility of vertically aligned multiwalled carbon nanotubes (MWCNT), used as nanomodification to optimize the properties of prostheses-embedded microelectrodes that induce electrical stimulation of surviving retinal cells. Methods. MWCNT were synthesized on silicon wafers. Their growth was achieved by iron particles (Fe) or mixtures of iron-platinum (Fe-Pt) and iron-titanium (Fe-Ti) acting as catalysts. Viability, growth, adhesion, and gene expression of L-929 and retinal precursor (R28) cells were analyzed after nondirect and direct contact. Results. Nondirect contact had almost no influence on cell growth, as measured in comparison to reference materials with defined levels of cytotoxicity. Both cell types exhibited good proliferation properties on each MWCNT-coated wafer. Viability ranged from 95.9 to 99.8%, in which better survival was observed for nonfunctionalized MWCNT generated with the Fe-Pt and Fe-Ti catalyst mixtures. R28 cells grown on the MWCNT-coated wafers showed a decreased gene expression associated with neural and glial properties. Expression of the cell cycle-related genes CCNC, MYC, and TP53 was slightly downregulated. Cultivation on plasma-treated MWCNT did not lead to additional changes. Conclusions. All tested MWCNT-covered slices showed good biocompatibility profiles, confirming that this nanotechnology is a promising tool to improve prostheses bearing electrodes which connect with retinal tissue. PMID:27200182

  1. Human embryonic stem cells and embryonal carcinoma cells have overlapping and distinct metabolic signatures.

    PubMed

    Abu Dawud, Raed; Schreiber, Kerstin; Schomburg, Dietmar; Adjaye, James

    2012-01-01

    While human embryonic stem cells (hESCs) and human embryonal carcinoma cells (hECCs) have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS). Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline) or lower (e.g., glutamic acid, mannitol, malic acid, GABA) in hESCs (H9) compared to hECCs (NTERA2), these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O(2) while oxidative phosphorylation (OXPHOS) is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency. PMID:22768158

  2. Human embryonic stem cells and embryonal carcinoma cells have overlapping and distinct metabolic signatures.

    PubMed

    Abu Dawud, Raed; Schreiber, Kerstin; Schomburg, Dietmar; Adjaye, James

    2012-01-01

    While human embryonic stem cells (hESCs) and human embryonal carcinoma cells (hECCs) have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS). Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline) or lower (e.g., glutamic acid, mannitol, malic acid, GABA) in hESCs (H9) compared to hECCs (NTERA2), these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O(2) while oxidative phosphorylation (OXPHOS) is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency.

  3. Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin

    PubMed Central

    Diaz Perez, Silvia V.; Kim, Rachel; Li, Ziwei; Marquez, Victor E.; Patel, Sanjeet; Plath, Kathrin; Clark, Amander T.

    2012-01-01

    Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1–UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state. PMID:22058289

  4. Factors derived from Escherichia coli Nissle 1917, grown in different growth media, enhance cell death in a model of 5-fluorouracil-induced Caco-2 intestinal epithelial cell damage.

    PubMed

    Wang, Hanru; Bastian, Susan E P; Lawrence, Andrew; Howarth, Gordon S

    2015-01-01

    We evaluated supernatants (SNs) from Escherichia coli Nissle 1917 (EcN) grown in commonly used growth media for their capacity to affect the viability of Caco-2 colon cancer cells in the presence and absence of 5-Fluorouracil (5-FU) chemotherapy. EcN was grown in Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17). Human Caco-2 colon cancer cells were treated with DMEM (control), growth media alone (LB, TSB, MRS, and M17) or EcN SNs derived from these 4 media, in the presence and absence of 5-FU. Cell viability, reactive oxygen species (ROS), and cell monolayer permeability were determined. EcN SN in LB medium reduced Caco-2 cell viability significantly, to 51% at 48 h. The combination of this EcN SN and 5-FU further reduced cell viability to 37% at 48 h, compared to 5-FU control. MRS broth and EcN SN in MRS, together with 5-FU, generated significantly lower levels of ROS compared to 5-FU control. However, all 5-FU treatments significantly disrupted the Caco-2 cell barrier compared to control; with no significant differences observed among any of the 5-FU treatments. EcN SNs (LB+) was most effective at decreasing the viability of Caco-2 cells. This could indicate a potential role for this EcN SN in chemoprevention for colon cancer. PMID:25625670

  5. Factors derived from Escherichia coli Nissle 1917, grown in different growth media, enhance cell death in a model of 5-fluorouracil-induced Caco-2 intestinal epithelial cell damage.

    PubMed

    Wang, Hanru; Bastian, Susan E P; Lawrence, Andrew; Howarth, Gordon S

    2015-01-01

    We evaluated supernatants (SNs) from Escherichia coli Nissle 1917 (EcN) grown in commonly used growth media for their capacity to affect the viability of Caco-2 colon cancer cells in the presence and absence of 5-Fluorouracil (5-FU) chemotherapy. EcN was grown in Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17). Human Caco-2 colon cancer cells were treated with DMEM (control), growth media alone (LB, TSB, MRS, and M17) or EcN SNs derived from these 4 media, in the presence and absence of 5-FU. Cell viability, reactive oxygen species (ROS), and cell monolayer permeability were determined. EcN SN in LB medium reduced Caco-2 cell viability significantly, to 51% at 48 h. The combination of this EcN SN and 5-FU further reduced cell viability to 37% at 48 h, compared to 5-FU control. MRS broth and EcN SN in MRS, together with 5-FU, generated significantly lower levels of ROS compared to 5-FU control. However, all 5-FU treatments significantly disrupted the Caco-2 cell barrier compared to control; with no significant differences observed among any of the 5-FU treatments. EcN SNs (LB+) was most effective at decreasing the viability of Caco-2 cells. This could indicate a potential role for this EcN SN in chemoprevention for colon cancer.

  6. Human Embryonic Stem Cell Lines with Lesions in FOXP3 and NF1

    PubMed Central

    Zhu, Hui; Behr, Barry; Reddy, Vikrant V.; Hughes, Mark; Pan, Yuqiong; Baker, Julie

    2016-01-01

    Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of blastocyst staged embryos. Spare blastocyst staged embryos were obtained by in vitro fertilization (IVF) and donated for research purposes. hESCs carrying specific mutations can be used as a powerful cell system in modeling human genetic disorders. We obtained preimplantation genetic diagnosed (PGD) blastocyst staged embryos with genetic mutations that cause human disorders and derived hESCs from these embryos. We applied laser assisted micromanipulation to isolate the inner cell mass from the blastocysts and plated the ICM onto the mouse embryonic fibroblast cells. Two hESC lines with lesions in FOXP3 and NF1 were established. Both lines maintain a typical undifferentiated hESCs phenotype and present a normal karyotype. The two lines express a panel of pluripotency markers and have the potential to differentiate to the three germ layers in vitro and in vivo. The hESC lines with lesions in FOXP3 and NF1 are available for the scientific community and may serve as an important resource for research into these disease states. PMID:26990425

  7. Structural Complexity of Non-acid Glycosphingolipids in Human Embryonic Stem Cells Grown under Feeder-free Conditions*

    PubMed Central

    Barone, Angela; Benktander, John; Ångström, Jonas; Aspegren, Anders; Björquist, Petter; Teneberg, Susann; Breimer, Michael. E.

    2013-01-01

    Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 109 cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Lex pentaosylceramide, and the Ley hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated. PMID:23404501

  8. Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale.

    PubMed

    Cho, Myung-Soo; Hwang, Dong-Youn; Kim, Dong-Wook

    2008-01-01

    Cell-replacement therapy using human embryonic stem cells (hESCs) holds great promise in treating Parkinson's disease. We have recently reported a highly efficient method to generate functional dopaminergic (DA) neurons from hESCs. Our method includes a unique step, the formation of spherical neural masses (SNMs), and offers the highest yield of DA neurons ever achieved so far. In this report, we describe our method step by step, covering not only how to differentiate hESCs into DA neurons at a high yield, but also how to amplify, freeze and thaw the SNMs, which are the key structures that make our protocol unique and advantageous. Although the whole process of generation of DA neurons from hESCs takes about 2 months, only 14 d are needed to derive DA neurons from the SNMs.

  9. Effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract

    SciTech Connect

    Bajpai, P.; Margaritis, A.

    1987-01-01

    The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25-35 degrees C, but dropped sharply beyond 35 degrees C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35 degrees C. However, with the immobilized cells, ethanol yield remained almost constant in the temperatue range 25-45 degrees C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40 degrees C. For the pH range between 3 and 7, the free-cell optimum for growth and product formation was found to be circa pH 5. At this pH, the specific growth rate was 0.35/h and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6. (Refs. 22).

  10. Differing Lectin Binding Profiles among Human Embryonic Stem Cells and Derivatives Aid in the Isolation of Neural Progenitor Cells

    PubMed Central

    Dodla, Mahesh C.; Young, Amber; Venable, Alison; Hasneen, Kowser; Rao, Raj R.; Machacek, David W.; Stice, Steven L.

    2011-01-01

    Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells. PMID:21850265

  11. Concerns about eroding the ethical barrier to in vitro eugenics: lessons from the hESC debate.

    PubMed

    Pugh, Jonathan

    2014-11-01

    In his discussion of in vitrogametogenesis, Rob Sparrow claims that an ethical barrier to development of this technology is that many jurisdictions currently prohibit the practice of creating embryos solely for the purpose of research. However, he suggests that this ethical barrier will soon be eroded, in view of the fact that in vitro gametogenesis could serve as a powerful new technology to overcome infertility. In this commentary, I argue that Sparrow is being overly optimistic in his analysis here. I claim that the debate over so-called compromise positions in the human embryonic stem cell debate suggests that the purpose of the research for which a research embryo is created is unlikely to be considered as having any significant bearing on the moral permissibility of the practice for those who oppose it. Even though in vitro gametogenesis could serve as a powerful new technology to overcome infertility, I argue that opponents of the practice of creating embryos solely for research purposes would still view the creation of research embryos that the development of in vitro gametogenesis would require, as being incompatible with affording the embryo proper moral respect. I conclude by suggesting that Sparrow's analysis of the potential benefits of in vitro gametogenesis provides us with further reasons to scrutinise the unconvincing arguments that are often cited in favour of prohibiting the practice of creating embryos solely for research purposes.

  12. SCL/TAL1-mediated Transcriptional Network Enhances Megakaryocytic Specification of Human Embryonic Stem Cells

    PubMed Central

    Toscano, Miguel G; Navarro-Montero, Oscar; Ayllon, Veronica; Ramos-Mejia, Veronica; Guerrero-Carreno, Xiomara; Bueno, Clara; Romero, Tamara; Lamolda, Mar; Cobo, Marien; Martin, Francisco; Menendez, Pablo; Real, Pedro J

    2015-01-01

    Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34+ progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs. PMID:25292191

  13. A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells

    PubMed Central

    Zhu, Zengrong; Verma, Nipun; González, Federico; Shi, Zhong-Dong; Huangfu, Danwei

    2015-01-01

    Summary The development of new gene-editing tools, in particular the CRISPR/Cas system, has greatly facilitated site-specific mutagenesis in human embryonic stem cells (hESCs), including the introduction or correction of patient-specific mutations for disease modeling. However, integration of a reporter gene into an endogenous locus in hESCs still requires a lengthy and laborious two-step strategy that involves first drug selection to identify correctly targeted clones and then excision of the drug-resistance cassette. Through the use of iCRISPR, an efficient gene-editing platform we recently developed, this study demonstrates a knockin strategy without drug selection for both active and silent genes in hESCs. Lineage-specific hESC reporter lines are useful for real-time monitoring of cell-fate decisions and lineage tracing, as well as enrichment of specific cell populations during hESC differentiation. Thus, this selection-free knockin strategy is expected to greatly facilitate the use of hESCs for developmental studies, disease modeling, and cell-replacement therapy. PMID:26028531

  14. Lipid content and fatty acid composition of green algae Scenedesmus obliquus grown in a constant cell density apparatus

    NASA Technical Reports Server (NTRS)

    Choi, K. J.; Nakhost, Z.; Barzana, E.; Karel, M.

    1987-01-01

    The lipids of alga Scenedesmus obliquus grown under controlled conditions were separated and fractionated by column and thin-layer chromatography, and fatty acid composition of each lipid component was studied by gas-liquid chromatography (GLC). Total lipids were 11.17%, and neutral lipid, glycolipid and phospholipid fractions were 7.24%, 2.45% and 1.48% on a dry weight basis, respectively. The major neutral lipids were diglycerides, triglycerides, free sterols, hydrocarbons and sterol esters. The glycolipids were: monogalactosyl diglyceride, digalactosyl diglyceride, esterified sterol glycoside, and sterol glycoside. The phospholipids included: phosphatidyl choline, phosphatidyl glycerol and phosphatidyl ethanolamine. Fourteen fatty acids were identified in the four lipid fractions by GLC. The main fatty acids were C18:2, C16:0, C18:3(alpha), C18:1, C16:3, C16:1, and C16:4. Total unsaturated fatty acid and essential fatty acid compositions of the total algal lipids were 80% and 38%, respectively.

  15. Growing Stem Cells: The Impact of Federal Funding Policy on the U.S. Scientific Frontier

    ERIC Educational Resources Information Center

    Furman, Jeffrey L.; Murray, Fiona; Stern, Scott

    2012-01-01

    This paper articulates a citation-based approach to science policy evaluation and employs that approach to investigate the impact of the United States' 2001 policy regarding the federal funding of human embryonic stem cell (hESC) research. We evaluate the impact of the policy on the level of U.S. hESC research, the U.S. position at the knowledge…

  16. Stability of single and tandem junction a-Si:H solar cells grown using the ECR process

    SciTech Connect

    Dalal, V.L.; Maxson, T.; Girvan, R.; Haroon, S.

    1997-07-01

    The authors report on the fabrication and stability tests of single junction a-Si:H, and tandem junction a-Si:H/A-Si:H solar cells using the ECR process under high hydrogen dilution (H-ECR process). They show that devices with high fill factors can be made using the H-ECR process. They also report on the stability studies of the solar cells under 1 and 2-sun illumination conditions. The solar cells show very little degradation even after 500 hours of illumination under 2 x sunlight illumination.

  17. Differences in excitation energy transfer of Arthrospira platensis cells grown in seawater medium and freshwater medium, probed by time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Arba, Muhammad; Aikawa, Shimpei; Niki, Kenta; Yokono, Makio; Kondo, Akihiko; Akimoto, Seiji

    2013-11-01

    Excitation energy transfer of Arthrospira platensis cells grown in f/2 medium (a high salinity medium) and SOT medium (a control) was investigated by steady-state and time-resolved spectroscopies. Growth in f/2 medium induced changes in absorption and fluorescence spectra as well as in the energy transfer pathways. Excitation energy captured by phycobilisome (PBS) was transferred directly to photosystem (PS) I, instead of being first transferred to an intermediate (PBS → PSII → PSI), as observed in SOT medium. The respiration rate increased while photosynthetic rate reduced in f/2 medium. Possible causes of the differences in light-harvesting and energy-transfer processes between the two media are discussed.

  18. Effects of the aspect ratio on the dye adsorption of ZnO nanorods grown by using a sonochemical method for dye-sensitized solar cells

    NASA Astrophysics Data System (ADS)

    Choi, Seok Cheol; Yun, Won Suk; Sohn, Sang Ho; Oh, Sang Jin

    2012-11-01

    Well-aligned ZnO nanorods for the photoelectrode of dye-sensitized solar cells (DSSCs) were grown via a sonochemical method, and the effects of their aspect ratios on the dye adsorption in DSSCs were studied. The control of the aspect ratio of well-aligned ZnO nanorods was performed by tuning the mole concentration of zinc acetate dehydrate in the range of 0.04-0.06M. The dye amounts adsorbed in the ZnO nanorods were estimated from the UV-Visible absorbance by using the Beer-Lambert law. The efficiency of DSSCs with ZnO nanorods was measured to investigate the effects of the aspect ratio of the ZnO nanorods on the dye adsorption properties. A change in the aspect ratio of the ZnO nanorods was founded to yield a change in their dye adsorption ability, resulting in a change in the efficiency of the DSSCs.

  19. The light intensity under which cells are grown controls the type of peripheral light-harvesting complexes that are assembled in a purple photosynthetic bacterium.

    PubMed

    Brotosudarmo, Tatas H P; Collins, Aaron M; Gall, Andrew; Roszak, Aleksander W; Gardiner, Alastair T; Blankenship, Robert E; Cogdell, Richard J

    2011-11-15

    The differing composition of LH2 (peripheral light-harvesting) complexes present in Rhodopseudomonas palustris 2.1.6 have been investigated when cells are grown under progressively decreasing light intensity. Detailed analysis of their absorption spectra reveals that there must be more than two types of LH2 complexes present. Purified HL (high-light) and LL (low-light) LH2 complexes have mixed apoprotein compositions. The HL complexes contain PucAB(a) and PucAB(b) apoproteins. The LL complexes contain PucAB(a), PucAB(d) and PucB(b)-only apoproteins. This mixed apoprotein composition can explain their resonance Raman spectra. Crystallographic studies and molecular sieve chromatography suggest that both the HL and the LL complexes are nonameric. Furthermore, the electron-density maps do not support the existence of an additional Bchl (bacteriochlorophyll) molecule; rather the density is attributed to the N-termini of the α-polypeptide.

  20. The light intensity under which cells are grown controls the type of peripheral light-harvesting complexes that are assembled in a purple photosynthetic bacterium

    SciTech Connect

    Brotosudarmo, Tatas H. P.; Collins, Aaron M.; Gall, Andrew; Roszak, Aleksander W.; Gardiner, Alastair T.; Blankenship, Robert E.; Cogdell, Richard J.

    2011-11-15

    The differing composition of LH2 (peripheral light-harvesting) complexes present in Rhodopseudomonas palustris 2.1.6 have been investigated when cells are grown under progressively decreasing light intensity. Analysis of the absorption spectra reveals there must be more than two types of LH2 complexes present. Purified HL (high-light) and LL (low-light) LH2 complexes have mixed apoprotein compositions. The HL complexes contain PucABa and PucABb apoproteins. The LL complexes contain PucABa, PucABd and PucBb-only apoproteins. This mixed apoprotein composition can explain their resonance Raman spectra.

  1. miR-200c and GATA binding protein 4 regulate human embryonic stem cell renewal and differentiation.

    PubMed

    Huang, Hsiao-Ning; Chen, Shao-Yin; Hwang, Shiaw-Min; Yu, Ching-Chia; Su, Ming-Wei; Mai, Wei; Wang, Hsei-Wei; Cheng, Wei-Chung; Schuyler, Scott C; Ma, Nianhan; Lu, Frank Leigh; Lu, Jean

    2014-03-01

    Human embryonic stem cells (hESCs) are functionally unique for their self-renewal ability and pluripotency, but the molecular mechanisms giving rise to these properties are not fully understood. hESCs can differentiate into embryoid bodies (EBs) containing ectoderm, mesoderm, and endoderm. In the miR-200 family, miR-200c was especially enriched in undifferentiated hESCs and significantly downregulated in EBs. The knockdown of the miR-200c in hESCs downregulated Nanog expression, upregulated GATA binding protein 4 (GATA4) expression, and induced hESC apoptosis. The knockdown of GATA4 rescued hESC apoptosis induced by downregulation of miR-200c. miR-200c directly targeted the 3'-untranslated region of GATA4. Interestingly, the downregulation of GATA4 significantly inhibited EB formation in hESCs. Overexpression of miR-200c inhibited EB formation and repressed the expression of ectoderm, endoderm, and mesoderm markers, which could partially be rescued by ectopic expression of GATA4. Fibroblast growth factor (FGF) and activin A/nodal can sustain hESC renewal in the absence of feeder layer. Inhibition of transforming growth factor-β (TGF-β[Symbol: see text])/activin A/nodal signaling by SB431542 treatment downregulated the expression of miR-200c. Overexpression of miR-200c partially rescued the expression of Nanog/phospho-Smad2 that was downregulated by SB431542 treatment. Our observations have uncovered novel functions of miR-200c and GATA4 in regulating hESC renewal and differentiation.

  2. Effects of radiation of InP cells epitaxially grown on Si and GaAs substrates

    NASA Technical Reports Server (NTRS)

    Weinberg, I.; Swartz, C. K.; Brinker, D. J.; Wilt, D. M.

    1990-01-01

    The properties of heteroepitaxial InP cells were determined both before and after 10-MeV proton irradiations. Numerical values, obtained for the diffusion and recombination components of the reverse saturation currents, were found to be consistent with the distribution of dislocations. The radiation resistance of the heteroepitaxial cells was significantly greater than that observed for n/p homoepitaxial InP cells. The carrier removal rate, obtained by C-V measurements, was 1800/cm for 10-MeV protons compared with 2.2/cm for 1-MeV electrons. The high carrier removal rate was found to have no significant effect on the cell's series resistance. It was concluded that the heteroepitaxial cell performance is dominated by the high dislocation density attributable to lattice constant mismatch. Although the efficiencies of the present cells are low, the recent achievement of 13.7 percent AM0 efficiencies using a GaAs substrate demonstrates the marked improvement that can be attained using more appropriate transition layers.

  3. Differences In Early T-Cell Signaling In Cultures Grown In a Rotating Clinostat vs. Static Controls

    NASA Technical Reports Server (NTRS)

    Alexamder. M.; Nelman-Gonzales, M.; Penkala, J.; Sams, C.

    1999-01-01

    Altered gravity has previously been demonstrated to be a stress that can influence components of the immune system. Specifically, T-cell activation has been shown to be affected by changes in gravity, exhibiting a decrease in proliferative response to in vitro stimulation in microgravity. Subsequent ground based studies utilizing a rotating clinostat to model some of the effects of microgravity have been consistent with earlier flight based experiments. These ground and flight experiments have examined T-cell activation by measuring various responses including production of cytokines, DNA synthesis and the production of various cell surface activation markers. These indicators of T-cell activation were measured anywhere from 4 to 72 hours after stimulation. Prior to the work described here, the initial signaling events in T-cell activation had not been directly examined. The goal of this project was to determine how the process of early signal transduction was affected by growth in a rotating clinostat. Here we directly show a defect in signaling from TCR to MAPK in purified peripheral T-cells activated in the clinostat by OKT3/antiCD28 coated microbeads as compared to static controls.

  4. Lysophosphatidic acid and sphingosine 1-phosphate metabolic pathways and their receptors are differentially regulated during decidualization of human endometrial stromal cells.

    PubMed

    Brünnert, D; Sztachelska, M; Bornkessel, F; Treder, N; Wolczynski, S; Goyal, P; Zygmunt, M

    2014-10-01

    In the luteal phase, human endometrial stromal cells (HESCs) undergo proliferation, migration and differentiation during the decidualization process under the control of the ovarian steroids progesterone and estrogen. Proper decidualization of stromal cells is required for blastocyst implantation and the development of pregnancy. The proliferation, migration and differentiation of HESCs in decidualization do not require the presence of a blastocyst but are greatly accelerated during implantation. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are potent bioactive lysophospholipids that have critical roles in various physiological and pathophysiological processes, including inflammation, angiogenesis and cancer. The expression of the enzymes involved in LPA and S1P turnover and their receptors in HESCs during decidualization has not been characterized yet. We found that the LPAR1 and LPAR6 and S1PR3 receptors are highly expressed in HESCs. LPAR1, autotaxin (ATX), an LPA producing enzyme and lipid phosphate phosphatase 3 were up-regulated during decidualization. Interestingly, the expression of all S1P receptor subtypes and LPA receptors (LPAR2-6) mRNA was down-regulated after decidualization. We found that SPHK1 is highly expressed in HESCs, and is up-regulated during decidualization. S1P phosphatase SGPP1 and S1P lyase SGPL1 are highly expressed in HESCs. SGPP1 mRNA expression was significantly up-regulated in decidualized HESCs. In conclusion, this study shows the first time that specific LPA and S1P receptors and their metabolizing enzymes are highly regulated in HESCs during decidualization. Furthermore, we suggest that LPAR1 receptor-mediated signaling in HESCs may be crucial in decidualization process. SPHK1 activity and high turnover of S1P and LPA might be essential for precise regulation of their signaling during decidualization of human endometrium and implantation. PMID:24994816

  5. Transcriptional activation of human CDCA8 gene regulated by transcription factor NF-Y in embryonic stem cells and cancer cells.

    PubMed

    Dai, Can; Miao, Cong-Xiu; Xu, Xiao-Ming; Liu, Lv-Jun; Gu, Yi-Fan; Zhou, Di; Chen, Lian-Sheng; Lin, Ge; Lu, Guang-Xiu

    2015-09-11

    The cell division cycle associated 8 (CDCA8) gene plays an important role in mitosis. Overexpression of CDCA8 was reported in some human cancers and is required for cancer growth and progression. We found CDCA8 expression was also high in human ES cells (hESCs) but dropped significantly upon hESC differentiation. However, the regulation of CDCA8 expression has not yet been studied. Here, we characterized the CDCA8 promoter and identified its cis-elements and transcription factors. Three transcription start sites were identified. Reporter gene assays revealed that the CDCA8 promoter was activated in hESCs and cancer cell lines. The promoter drove the reporter expression specifically to pluripotent cells during early mouse embryo development and to tumor tissues in tumor-bearing mice. These results indicate that CDCA8 is transcriptionally activated in hESCs and cancer cells. Mechanistically, two key activation elements, bound by transcription factor NF-Y and CREB1, respectively, were identified in the CDCA8 basic promoter by mutation analyses and electrophoretic motility shift assays. NF-Y binding is positively correlated with promoter activities in different cell types. Interestingly, the NF-YA subunit, binding to the promoter, is primarily a short isoform in hESCs and a long isoform in cancer cells, indicating a different activation mechanism of the CDCA8 transcription between hESCs and cancer cells. Finally, enhanced CDCA8 promoter activities by NF-Y overexpression and reduced CDCA8 transcription by NF-Y knockdown further verified that NF-Y is a positive regulator of CDCA8 transcription. Our study unearths the molecular mechanisms underlying the activation of CDCA8 expression in hESCs and cancer cells, which provides a better understanding of its biological functions.

  6. Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors

    PubMed Central

    Salomonis, Nathan; Nelson, Brandon; Vranizan, Karen; Pico, Alexander R.; Hanspers, Kristina; Kuchinsky, Allan; Ta, Linda; Mercola, Mark; Conklin, Bruce R.

    2009-01-01

    The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation. PMID:19893621

  7. Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells.

    PubMed

    Duggal, Galbha; Warrier, Sharat; Ghimire, Sabitri; Broekaert, Dorien; Van der Jeught, Margot; Lierman, Sylvie; Deroo, Tom; Peelman, Luc; Van Soom, Ann; Cornelissen, Ria; Menten, Björn; Mestdagh, Pieter; Vandesompele, Jo; Roost, Matthias; Slieker, Roderick C; Heijmans, Bastiaan T; Deforce, Dieter; De Sutter, Petra; De Sousa Lopes, Susana Chuva; Heindryckx, Björn

    2015-09-01

    Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation.

  8. High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling

    PubMed Central

    Fu, Xin; Xie, Fang-Nan; Dong, Ping; Li, Qiu-Chen; Yu, Guang-Yan; Xiao, Ran

    2016-01-01

    Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However, insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs), which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore, we investigated the effects of sodium fluoride (NaF) on the proliferation, differentiation and viability of H9 hESCs. For the first time, we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology, mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway, coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a p-JNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs. PMID:26859149

  9. Upregulation of cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on magnesium alloy surface coating with tricalcium phosphate.

    PubMed

    Jiang, Tianlong; Guo, Lei; Ni, Shenghui; Zhao, Yuyan

    2015-04-01

    Magnesium (Mg) alloys have been demonstrated to be viable orthopedic implants because of mechanical and biocompatible properties similar to natural bone. In order to improve its osteogenic properties, a porous β-tricalcium phosphate (β-TCP) was coated on the Mg-3AI-1Zn alloy by alkali-heat treatment technique. The human bone-derived cells (SaOS-2) were cultured on (β-TCP)-Mg-3AI-1Zn in vitro, and the osteoblast response, the morphology and the elements on this alloy surface were investigated. Also, the regulation of key intracellular signalling proteins was investigated in the SaOS-2 cells cultured on alloy surface. The results from scanning electron microscope and immunofluorescence staining demonstrated that (β-TCP)-Mg-3AI-1Zn induced significant osteogenesis. SaOS-2 cell proliferation was improved by β-TCP coating. Moreover, the (β-TCP)-Mg-3AI-1Zn surface induced activation of key intracellular signalling proteins in SaOS-2 cells. We observed an enhanced activation of Src homology and collagen (Shc), a common point of integration between bone morphogenetic protein 2, and the Ras/mitogen-activated protein kinase (MAPK) pathway. ERK1/2 MAP kinase activation was also upregulated, suggesting a role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos (member of the activated protein-1) was also shown to be upregulated in osteoblasts cultured on the (β-TCP)-Mg-3AI-1Zn. These results suggest that β-TCP coating may contribute to successful osteoblast function on Mg alloy surface. (β-TCP)-Mg-3AI-1Zn may upregulate cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on Mg alloy surface.

  10. Does vector-free gravity simulate microgravity? Functional and morphologic attributes of clinorotated nerve and muscle grown in cell culture

    NASA Technical Reports Server (NTRS)

    Gruener, R.; Hoeger, G.

    1988-01-01

    Cocultured Xenopus neurons and myocytes were subjected to non-vectorial gravity by clinostat rotation to determine if microgravity, during space flights, may affect cell development and communications. Clinorotated cells showed changes consistent with the hypothesis that cell differentiation, in microgravity, is altered by interference with cytoskeleton-related mechanisms. We found: increases in the myocyte and its nuclear area, "fragmentation" of nucleoli, appearance of neuritic "aneurysms", decreased growth in the presence of "trophic" factors, and decreased yolk utilization. The effects were most notable at 1-10 rpm and depended on the onset and duration of rotation. Some parameters returned to near control values within 48 hrs after cessation of rotation. Cells from cultures rotated at higher speeds (>50 rpm) appeared comparable to controls. Compensation by centrifugal forces may account for this finding. Our data are consistent, in principle, with effects on other, flighted cells and suggest that "vector-free" gravity may simulate certain aspects of microgravity. The distribution of acetylcholine receptor aggregates, on myocytes, was also altered. This indicates that brain development, in microgravity, may also be affected.

  11. HIGH-THROUGHPUT SCREENING ASSAY FOR THE IDENTIFICATION OF COMPOUNDS REGULATING SELF-RENEWAL AND DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS

    PubMed Central

    Desbordes, Sabrina C.; Placantonakis, Dimitris G.; Ciro, Anthony; Socci, Nicholas D.; Lee, Gabsang; Djaballah, Hakim; Studer, Lorenz

    2009-01-01

    Summary High-throughput screening (HTS) of chemical libraries has become a critical tool in basic biology and drug discovery. However, its implementation and the adaptation of high content assays to human embryonic stem cells (hESCs) have been hampered by multiple technical challenges. Here we present a strategy to adapt hESCs to HTS conditions, resulting in an assay suitable for the discovery of small molecules that drive hESC self-renewal or differentiation. Use of this new assay has led to the identification of several marketed drugs and natural compounds promoting short-term hESC maintenance and compounds directing early lineage choice during differentiation. Global gene expression analysis upon drug treatment defines known and novel pathways correlated to hESC self-renewal and differentiation. Our results demonstrate feasibility of hESC-based HTS and enhance the repertoire of chemical compounds for manipulating hESC fate. The availability of high content assays should accelerate progress in basic and translational hESC biology. PMID:18522853

  12. Comparative Analysis of Whole-Genome Gene Expression Changes in Cultured Human Embryonic Stem Cells in Response to Low, Clinical Diagnostic Relevant, and High Doses of Ionizing Radiation Exposure.

    PubMed

    Sokolov, Mykyta; Nguyen, Van; Neumann, Ronald

    2015-01-01

    The biological effects of low-dose ionizing radiation (LDIR) exposure in humans are not comprehensively understood, generating a high degree of controversy in published literature. The earliest stages of human development are known to be among the most sensitive to stress exposures, especially genotoxic stresses. However, the risks stemming from exposure to LDIR, particularly within the clinical diagnostic relevant dose range, have not been directly evaluated in human embryonic stem cells (hESCs). Here, we describe the dynamics of the whole genome transcriptional responses of different hESC lines to both LDIR and, as a reference, high-dose IR (HDIR). We found that even doses as low as 0.05 Gy could trigger statistically significant transient changes in a rather limited subset of genes in all hESCs lines examined. Gene expression signatures of hESCs exposed to IR appear to be highly dose-, time-, and cell line-dependent. We identified 50 genes constituting consensus gene expression signature as an early response to HDIR across all lines of hESC examined. We observed substantial differences in biological pathways affected by either LDIR or HDIR in hESCs, suggesting that the molecular mechanisms underpinning the responses of hESC may fundamentally differ depending on radiation doses. PMID:26133243

  13. Comparison of single junction AlGaInP and GaInP solar cells grown by molecular beam epitaxy

    SciTech Connect

    Masuda, Taizo Tomasulo, Stephanie; Lang, Jordan R.; Lee, Minjoo Larry

    2015-03-07

    We have investigated ∼2.0 eV (Al{sub x}Ga{sub 1−x}){sub 0.51}In{sub 0.49}P and ∼1.9 eV Ga{sub 0.51}In{sub 0.49}P single junction solar cells grown on both on-axis and misoriented GaAs substrates by molecular beam epitaxy (MBE). Although lattice-matched (Al{sub x}Ga{sub 1−x}){sub 0.51}In{sub 0.49}P solar cells are highly attractive for space and concentrator photovoltaics, there have been few reports on the MBE growth of such cells. In this work, we demonstrate open circuit voltages (V{sub oc}) ranging from 1.29 to 1.30 V for Ga{sub 0.51}In{sub 0.49}P cells, and 1.35–1.37 V for (Al{sub x}Ga{sub 1−x}){sub 0.51}In{sub 0.49}P cells. Growth on misoriented substrates enabled the bandgap-voltage offset (W{sub oc} = E{sub g}/q − V{sub oc}) of Ga{sub 0.51}In{sub 0.49}P cells to decrease from ∼575 mV to ∼565 mV, while that of (Al{sub x}Ga{sub 1−x}){sub 0.51}In{sub 0.49}P cells remained nearly constant at 620 mV. The constant W{sub oc} as a function of substrate offcut for (Al{sub x}Ga{sub 1−x}){sub 0.51}In{sub 0.49}P implies greater losses from non-radiative recombination compared with the Ga{sub 0.51}In{sub 0.49}P devices. In addition to larger W{sub oc} values, the (Al{sub x}Ga{sub 1−x}){sub 0.51}In{sub 0.49}P cells exhibited significantly lower internal quantum efficiency (IQE) values than Ga{sub 0.51}In{sub 0.49}P cells due to recombination at the emitter/window layer interface. A thin emitter design is experimentally shown to be highly effective in improving IQE, particularly at short wavelengths. Our work shows that with further optimization of both cell structure and growth conditions, MBE-grown (Al{sub x}Ga{sub 1−x}){sub 0.51}In{sub 0.49}P will be a promising wide-bandgap candidate material for high-efficiency, lattice-matched multi-junction solar cells.

  14. High-Quality Perovskite Films Grown with a Fast Solvent-Assisted Molecule Inserting Strategy for Highly Efficient and Stable Solar Cells.

    PubMed

    Yuan, Shuai; Qiu, Zhiwen; Gao, Chaomin; Zhang, Hailiang; Jiang, Yanan; Li, Cuncheng; Yu, Jinghua; Cao, Bingqiang

    2016-08-31

    The performance of organolead halide perovskites based solar cells has been enhanced dramatically due to the morphology control of the perovskite films. In this paper, we present a fast solvent-assisted molecule inserting (S-AMI) strategy to grow high-quality perovskite film, in which the methylammonium iodide/2-propanol (MAI/IPA) solution is spin-coated onto a dimethylformamide (DMF) wetted mixed lead halide (PbX2) precursor film. The DMF can help the inserting of MAI molecules into the PbX2 precursor film and provide a solvent environment to help the grain growth of the perovskite film. The perovskite film grown by the S-AMI approach shows large and well-oriented grains and long carrier lifetime due to the reduced grain boundary. Solar cells constructed with these perovskite films yield an average efficiency over 17% along with a high average fill factor of 80%. Moreover, these unsealed solar cell devices exhibit good stability in an ambient atmosphere. PMID:27526617

  15. High-Quality Perovskite Films Grown with a Fast Solvent-Assisted Molecule Inserting Strategy for Highly Efficient and Stable Solar Cells.

    PubMed

    Yuan, Shuai; Qiu, Zhiwen; Gao, Chaomin; Zhang, Hailiang; Jiang, Yanan; Li, Cuncheng; Yu, Jinghua; Cao, Bingqiang

    2016-08-31

    The performance of organolead halide perovskites based solar cells has been enhanced dramatically due to the morphology control of the perovskite films. In this paper, we present a fast solvent-assisted molecule inserting (S-AMI) strategy to grow high-quality perovskite film, in which the methylammonium iodide/2-propanol (MAI/IPA) solution is spin-coated onto a dimethylformamide (DMF) wetted mixed lead halide (PbX2) precursor film. The DMF can help the inserting of MAI molecules into the PbX2 precursor film and provide a solvent environment to help the grain growth of the perovskite film. The perovskite film grown by the S-AMI approach shows large and well-oriented grains and long carrier lifetime due to the reduced grain boundary. Solar cells constructed with these perovskite films yield an average efficiency over 17% along with a high average fill factor of 80%. Moreover, these unsealed solar cell devices exhibit good stability in an ambient atmosphere.

  16. Maslinic Acid, a Triterpene from Olive, Affects the Antioxidant and Mitochondrial Status of B16F10 Melanoma Cells Grown under Stressful Conditions

    PubMed Central

    Mokhtari, Khalida; Rufino-Palomares, Eva E.; Pérez-Jiménez, Amalia; Reyes-Zurita, Fernando J.; Figuera, Celeny; García-Salguero, Leticia; Medina, Pedro P.; Peragón, Juan; Lupiáñez, José A.

    2015-01-01

    Maslinic acid (MA) is a natural compound whose structure corresponds to a pentacyclic triterpene. It is abundant in the cuticular lipid layer of olives. MA has many biological and therapeutic properties related to health, including antitumor, anti-inflammatory, antimicrobial, antiparasitic, antihypertensive, and antioxidant activities. However, no studies have been performed to understand the molecular mechanism induced by this compound in melanoma cancer. The objective of this study was to examine the effect of MA in melanoma (B16F10) cells grown in the presence or absence of fetal bovine serum (FBS). We performed cell proliferation measurements, and the reactive oxygen species (ROS) measurements using dihydrorhodamine 123 (DHR 123) and activities of catalase, glucose 6-phosphate dehydrogenase, glutathione S-transferase, and superoxide dismutase. These changes were corroborated by expression assays. FBS absence reduced cell viability decreasing IC50 values of MA. The DHR 123 data showed an increase in the ROS level in the absence of FBS. Furthermore, MA had an antioxidant effect at lower assayed levels measured as DHR and antioxidant defense. However, at higher dosages MA induced cellular damage by apoptosis as seen in the results obtained. PMID:26236377

  17. Development of Scalable Culture Systems for Human Embryonic Stem Cells

    PubMed Central

    Azarin, Samira M.; Palecek, Sean P.

    2009-01-01

    The use of human pluripotent stem cells, including embryonic and induced pluripotent stem cells, in therapeutic applications will require the development of robust, scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs), but challenges specific to hESCs will also have to be addressed, including development of defined, humanized culture media and substrates, monitoring spontaneous differentiation and heterogeneity in the cultures, and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. PMID:20161686

  18. Bioactive metabolite production and stress-related hormones in Devil's claw cell suspension cultures grown in bioreactors.

    PubMed

    Georgiev, Milen; Ludwig-Müller, Jutta; Weber, Jost; Stancheva, Nina; Bley, Thomas

    2011-03-01

    In a previous report, we showed that cell cultures of Harpagophytum procumbens, a South African plant with high medicinal value, accumulate high amounts of anti-inflammatory phenylethanoid glycosides during cultivation in shake-flasks. The aim of the present study was to transfer the phenylethanoid biosynthetic process to a 3-L stirred tank reactor and a 1-L glass-column bioreactor (operated with pulsed aeration). We found that, with stepwise increases in aeration, the stirred tank reactor yielded similar productivities of verbascoside (the major phenylethanoid glycoside in the cells) to those reported for shake-flask cultures (55.68 vs. 54.78 mg verbascoside/L/day, respectively). Transfer in the pulse-aerated column reactor resulted in 165.42 mg verbascoside/L/day, one of the highest yields reported to date. Further, to evaluate the physiological status of the suspended cells in the bioreactors cultures, we examined their hormone levels and compared them to those of cells in shake-flask cultures. While indole-3-acetic acid levels did not differ significantly between the bioreactor and shake-flask cultures, there were considerable differences in their levels of abscisic, jasmonic, and salicylic acids. These results are discussed with respect to relative stress levels in the different cultivation systems. PMID:21104241

  19. Stem Cells Grown in Osteogenic Medium on PLGA, PLGA/HA, and Titanium Scaffolds for Surgical Applications

    PubMed Central

    Asti, Annalia; Gastaldi, Giulia; Dorati, Rossella; Saino, Enrica; Conti, Bice; Visai, Livia; Benazzo, Francesco

    2010-01-01

    Pluripotent adipose tissue-derived stem cells (hASCs) can differentiate into various mesodermal cell types such as osteoblasts, chondroblasts, and myoblasts. We isolated hASCs from subcutaneous adipose tissue during orthopaedic surgery and induced the osteogenic differentiation for 28 days on three different synthetic scaffolds such as polylactide-co-glycolide (PLGA), polylactide-co-glycolide/hydroxyapatite (PLGA/HA), and trabecular titanium scaffolds (Ti6Al4V). Pore size can influence certain criteria such as cell attachment, infiltration, and vascularization. The aim of this study was to investigate the performance of PLGA and PLGA/HA scaffolds with a higher porosity, ranging between 75% and 84%, with respect to Ti scaffolds but with smaller pore size, seeded with hASCs to develop a model that could be used in the treatment of bone defects and fractures. Osteogenesis was assessed by ELISA quantitation of extracellular matrix protein expression, von Kossa staining, X-ray microanalysis, and scanning electron microscopy. The higher amount of protein matrix on the Ti scaffold with respect to PLGA and PLGA/HA leads to the conclusion that not only the type of material but the structure significantly affects cell proliferation. PMID:21234383

  20. Role of surface-electrical properties on the cell-viability of carbon thin films grown in nanodomain morphology

    NASA Astrophysics Data System (ADS)

    Javid, Amjed; Kumar, Manish; Yoon, Seokyoung; Lee, Jung Heon; Tajima, Satomi; Hori, Masaru; Geon Han, Jeon

    2016-07-01

    Carbon thin films, having a combination of unique physical and chemical properties, exhibit an interesting biocompatibility and biological response to living entities. Here, the carbon films are developed in the morphology form of nano-domains with nanoscale inter-domain separations, tuned by plasma conditions in the facing target magnetron sputtering process. The wettability and surface energy are found to have a close relation to the inter-domain separations. The chemical structure of carbon films exhibited the relative enhancement of sp3 in comparison to sp2 with the increase of domain separations. The cell-viability of these films shows promising results for L929 mouse fibroblast and Saos-2 bone cells, when inter-domain separation is increased. Electrical conductivity and surface energy are identified to play the key role in different time-scales during the cell-proliferation process. The contribution from electrical conductivity is dominant in the beginning of the cultivation, whereas with the passage of time (~3-5 d) the surface energy takes control over conductivity to enhance the cell proliferation.

  1. Hap4p overexpression in glucose-grown Saccharomyces cerevisiae induces cells to enter a novel metabolic state

    PubMed Central

    Lascaris, Romeo; Bussemaker, Harmen J; Boorsma, André; Piper, Matt; van der Spek, Hans; Grivell, Les; Blom, Jolanda

    2003-01-01

    Background Metabolic and regulatory gene networks generally tend to be stable. However, we have recently shown that overexpression of the transcriptional activator Hap4p in yeast causes cells to move to a state characterized by increased respiratory activity. To understand why overexpression of HAP4 is able to override the signals that normally result in glucose repression of mitochondrial function, we analyzed in detail the changes that occur in these cells. Results Whole-genome expression profiling and fingerprinting of the regulatory activity network show that HAP4 overexpression provokes changes that also occur during the diauxic shift. Overexpression of HAP4, however, primarily acts on mitochondrial function and biogenesis. In fact, a number of nuclear genes encoding mitochondrial proteins are induced to a greater extent than in cells that have passed through a normal diauxic shift: in addition to genes required for mitochondrial energy conservation they include genes encoding mitochondrial ribosomal proteins. Conclusions We show that overproduction of a single nuclear transcription factor enables cells to move to a novel state that displays features typical of, but clearly not identical to, other derepressed states. PMID:12537548

  2. Role of surface-electrical properties on the cell-viability of carbon thin films grown in nanodomain morphology

    NASA Astrophysics Data System (ADS)

    Javid, Amjed; Kumar, Manish; Yoon, Seokyoung; Lee, Jung Heon; Tajima, Satomi; Hori, Masaru; Geon Han, Jeon

    2016-07-01

    Carbon thin films, having a combination of unique physical and chemical properties, exhibit an interesting biocompatibility and biological response to living entities. Here, the carbon films are developed in the morphology form of nano-domains with nanoscale inter-domain separations, tuned by plasma conditions in the facing target magnetron sputtering process. The wettability and surface energy are found to have a close relation to the inter-domain separations. The chemical structure of carbon films exhibited the relative enhancement of sp3 in comparison to sp2 with the increase of domain separations. The cell-viability of these films shows promising results for L929 mouse fibroblast and Saos-2 bone cells, when inter-domain separation is increased. Electrical conductivity and surface energy are identified to play the key role in different time-scales during the cell-proliferation process. The contribution from electrical conductivity is dominant in the beginning of the cultivation, whereas with the passage of time (~3–5 d) the surface energy takes control over conductivity to enhance the cell proliferation.

  3. Conversion of embryonic stem cells into pancreatic beta-cell surrogates guided by ontogeny.

    PubMed

    Lees, Justin G; Tuch, Bernard E

    2006-05-01

    Cellular therapies to treat Type 1 diabetes are being devised and the use of human embryonic stem cells (hESCs) offers a solution to the issue of supply, because hESCs can be maintained in a pluripotent state indefinitely. Furthermore, hESCs have advantages in terms of their plasticity and reduced immunogenicity. Several strategies that have so far been investigated indicate that hESCs are capable of differentiating into insulin producing beta-cell surrogates. However the efficiency of the differentiation procedures used is generally quite low and the cell populations derived are often highly heterogenous. A strategy that appears to have long term potential is to design differentiation procedures based on the ontogeny of the beta-cell. The focus of this strategy is to replicate signaling processes that are known to be involved in the maturation of a beta-cell. The earliest pancreatic progenitors found in the developing vertebrate fetus are produced via a process known as gastrulation and form part of the definitive endoderm germ layer. hESCs have recently been differentiated into definitive endoderm with high efficiency using a differentiation procedure that mimics the signaling that occurs during gastrulation and the formation of the definitive endoderm. Subsequent events during pancreas development involve a section of the definitive endoderm forming into pancreatic epithelium, which then branches into the pancreatic mesenchyme to form islet clusters of endocrine cells. A proportion of the endocrine precursor cells within islets develop into insulin producing beta-cells. The challenge currently is to design hESC differentiation procedures that mimic the combined events of these stages of beta-cell development.

  4. Development of high-bandgap AlGaInP solar cells grown by organometallic vapor-phase epitaxy

    DOE PAGES

    Perl, Emmett E.; Simon, John; Geisz, John F.; Olavarria, Waldo; Young, Michelle; Duda, Anna; Friedman, Daniel J.; Steiner, Myles A.

    2016-03-29

    AlGaInP solar cells with bandgaps between 1.9 and 2.2 eV are investigated for use in next-generation multijunction photovoltaic devices. This quaternary alloy is of great importance to the development of III-V solar cells with five or more junctions and for cells optimized for operation at elevated temperatures because of the high bandgaps required in these designs. In this work, we explore the conditions for the organometallic vapor-phase epitaxy growth of AlGaInP and study their effects on cell performance. Initial efforts focused on developing ~2.0-eV AlGaInP solar cells with a nominal aluminum composition of 12%. Under the direct spectrum at 1000more » W/m2 (AM1.5D), the best of these samples had an open-circuit voltage of 1.59 V, a bandgap-voltage offset of 440 mV, a fill factor of 88.0%, and an efficiency of 14.8%. We then varied the aluminum composition of the alloy from 0% to 24% and were able to tune the bandgap of the AlGaInP layers from ~1.9 to ~2.2 eV. Furthermore, while the samples with a higher aluminum composition exhibited a reduced quantum efficiency and increased bandgap-voltage offset, the bandgap-voltage offset remained at 500 mV or less, up to a bandgap of ~2.1 eV.« less

  5. A scalable label-free approach to separate human pluripotent cells from differentiated derivatives.

    PubMed

    Willoughby, N A; Bock, H; Hoeve, M A; Pells, S; Williams, C; McPhee, G; Freile, P; Choudhury, D; De Sousa, P A

    2016-01-01

    The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells. PMID:26858819

  6. Thin, high quality GaInP compositionally graded buffer layers grown at high growth rates for metamorphic III-V solar cell applications

    NASA Astrophysics Data System (ADS)

    Garcia, I.; France, R. M.; Geisz, J. F.; Simon, J.

    2014-05-01

    The metamorphic growth of lattice-mismatched materials has allowed optimizing the bandgap combination in multijunction solar cells for the solar spectrum under consideration. Buffer structures are used to accommodate the lattice-mismatch by introducing dislocations and relaxing the material in a controlled way. However, the metamorphic buffers typically involve significant growth time and material usage, which increases the cost of these solar cells. In this work, the thinning of buffer structures with continuously, linearly graded misfit is addressed with the goal of increasing the cost-effectiveness of metamorphic multijunction solar cells. The relaxation dynamics and quality of the buffer layers analyzed were assessed by in-situ stress measurements and ex-situ measurements of residual strain, threading dislocation density and surface roughness. Their ultimate quality has been tested using these buffers as templates for the growth of 1 eV Ga0.73In0.27As solar cells. The deleterious effect of thinning the grade layer of these buffer structures from 2 to 1 μm was investigated. It is shown that prompting the relaxation of the buffer by using a stepwise misfit jump at the beginning of the grade layer improves the quality of the thinned buffer structure. The residual threading dislocation density of the optimized thin buffers, grown at a high growth rate of 7 μm/h, is 3×106 cm-2, and solar cells on these buffers exhibit near-ideal carrier collection efficiency and a Voc of 0.62 V at 1-sun direct terrestrial spectrum.

  7. Exogenous supplementation of Activin A enhances germ cell differentiation of human embryonic stem cells.

    PubMed

    Duggal, Galbha; Heindryckx, Björn; Warrier, Sharat; Taelman, Jasin; Van der Jeught, Margot; Deforce, Dieter; Chuva de Sousa Lopes, Susana; De Sutter, Petra

    2015-05-01

    Human embryonic stem cells (hESCs) derived in the presence of Activin A (ActA) demonstrate an increased differentiation propensity toward the germ cell lineage. In addition, mouse epiblast stem cells and mouse epiblast-like cells are poised toward germ cell differentiation and are derived in the presence of ActA. We therefore investigated whether supplementation with ActA enhances in vitro hESC differentiation toward germ cell lineage. ActA up-regulated early primordial germ cell (PGC) genes STELLA/DPPA3 (developmental pluripotency associated 3) and tyrosine kinase receptor cKIT in both ActA-derived and standard-derived hESCs indicating its role in priming hESCs toward the PGC lineage. Indeed, ActA plus bone morphogenic protein 4 (BMP4) strongly increased germ cell differentiation potential of hESCs based on the high expression of late PGC markers DAZL (deleted in azoospermia-like) and VASA/DDX4 (DEAD-box polypeptide 4) at mRNA and protein level. Hence, the combination of ActA with BMP4 provides an additional boost for hESCs to develop into postmigratory germ cells. Together with increased VASA expression in the presence of ActA and BMP4, we also observed up-regulation of endoderm-specific genes GATA4 (GATA binding protein 4) and GATA6. Finally, we were able to further mature these in vitro-derived PGC-like cells (PGCLCs) by culturing them in in vitro maturation (IVM) medium, resulting in the formation of germ cell-like clusters and induction of meiotic gene expression. In conclusion, we demonstrate for the first time a synergism between ActA and BMP4 in facilitating germ cell-directed differentiation of hESCs, which is enhanced by extended culture in IVM medium, as shown by cytoplasmic VASA-expressing PGCLCs. We propose a novel relationship between the endoderm and germ cell lineage during hESC differentiation.

  8. Does vector-free gravity simulate microgravity? Functional and morphologic attributes of clinorotated nerve and muscle grown in cell culture

    NASA Technical Reports Server (NTRS)

    Gruener, Raphael; Hoeger, Glenn

    1988-01-01

    Cocultured Xenopus neurons and myocytes were subjected to nonvectorial gravity by clinostat rotation to determine the effects of microgravity on cell development and communications. Observed effects included increases in the myocyte and its nuclear area, fragmentation of nucleoli, the appearance of neuritic aneurysms, decreased growth in the presence of trophic factors, and decreased yolk utilization. These effects were most notable at 1-10 rpm and depended on the onset and duration of rotation. It is found that, in microgravity, cell differentiation is altered by interference with cytoskeleton-related mechanisms. It is suggested that the alteration of the distribution of acetylcholine receptor aggregates on myocytes which occurs might indicate that microgravity affects brain development.

  9. Effect of Increasing Doses of γ-Radiation on Bone Marrow Stromal Cells Grown on Smooth and Rough Titanium Surfaces

    PubMed Central

    Huang, Bo; Guang, Mengkai; Ye, Jun; Gong, Ping; Tang, Hua

    2015-01-01

    Radiation therapy for oral and maxillofacial tumors could damage bone marrow stromal cells (BMSCs) in jaw, which caused dental implant failure. However, how radiation affects BMSCs on SLA (sandblasted with large-grits, acid-etched) surfaces is still unknown. The aim of this study was to investigate effect of different dose of γ-radiation on BMSCs on SLA and PT (polished titanium) surfaces. Rat BMSCs were radiated with 2, 4, and 8 Gy γ-radiation and then seeded on both surfaces. Cell adhesion, spreading, and proliferation were tested. The osteogenesis and the adipogenesis ability were examined by Alizarin-Red and Oil-Red staining, respectively. Real-time PCR was performed to detect osteogenic (osteocalcin, OCN; runt-related transcription factor 2, Runx2) and adipogenic (peroxisome proliferator-activated receptor gamma, PPARγ) gene expression at days 7 and 14 postirradiation. Results showed that γ-radiation reduced cell proliferation, adhesion, spreading, and osteogenic differentiation. 2 Gy radiation promoted adipogenic differentiation, but it was significantly decreased when dosage reached 4 Gy. In conclusion, results suggest that γ-radiation influenced BMSCs behaviors in a dosage-dependent manner except adipogenic differentiation, low dose promoted it, and high dose inhibited it. This effect was influenced by surface characteristics, which may explain the different failure rate of various implants in patients after radiation. PMID:26257788

  10. Effects of green-synthesized silver nanoparticles on lung cancer cells in vitro and grown as xenograft tumors in vivo

    PubMed Central

    He, Yan; Du, Zhiyun; Ma, Shijing; Liu, Yue; Li, Dongli; Huang, Huarong; Jiang, Sen; Cheng, Shupeng; Wu, Wenjing; Zhang, Kun; Zheng, Xi

    2016-01-01

    Silver nanoparticles (AgNPs) have now been recognized as promising therapeutic molecules and are extending their use in cancer diagnosis and therapy. This study demonstrates for the first time the antitumor activity of green-synthesized AgNPs against lung cancer in vitro and in vivo. Cytotoxicity effect was explored on human lung cancer H1299 cells in vitro by MTT and trypan blue assays. Apoptosis was measured by morphological assessment, and nuclear factor-κB (NF-κB) transcriptional activity was determined by a luciferase reporter gene assay. The expressions of phosphorylated stat3, bcl-2, survivin, and caspase-3 were examined by Western blot analysis. AgNPs showed dose-dependent cytotoxicity and stimulation of apoptosis in H1299 cells. The effects on H1299 cells correlated well with the inhibition of NF-κB activity, a decrease in bcl-2, and an increase in caspase-3 and survivin expression. AgNPs significantly suppressed the H1299 tumor growth in a xenograft severe combined immunodeficient (SCID) mouse model. The results demonstrate the anticancer activities of AgNPs, suggesting that they may act as potential beneficial molecules in lung cancer chemoprevention and chemotherapy, especially for early-stage intervention. PMID:27217750

  11. Cartilage Repair Using Human Embryonic Stem Cell-Derived Chondroprogenitors

    PubMed Central

    Kapacee, Zoher; Peng, Jiang; Lu, Shibi; Lucas, Robert J.; Hardingham, Timothy E.

    2014-01-01

    In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling. The protocol was also applied successfully to two lines of induced pluripotent stem cells (iPSCs). Chondrogenic cells derived from hESCs were encapsulated in fibrin gel and implanted in osteochondral defects in the patella groove of nude rats, and cartilage repair was evaluated by histomorphology and immunocytochemistry. Genes associated with chondrogenesis were upregulated during the protocol, and pluripotency-related genes were downregulated. Aggregation of chondrogenic cells was accompanied by high expression of SOX9 and strong staining with Safranin O. Culture with PluriSln1 was lethal for hESCs but was tolerated by hESC chondrogenic cells, and no OCT4-positive cells were detected in hESC chondrogenic cells. iPSCs were also shown to generate chondroprogenitors in this protocol. Repaired tissue in the defect area implanted with hESC-derived chondrogenic cells was stained for collagen II with little collagen I, but negligible collagen II was observed in the fibrin-only controls. Viable human cells were detected in the repair tissue at 12 weeks. The results show that chondrogenic cells derived from hESCs, using a chemically defined culture system, when implanted in focal defects were able to promote cartilage repair. This is a first step in evaluating these cells for clinical application for the treatment of cartilage lesions. PMID:25273540

  12. Human embryonic stem cell science and policy: The case of Iran☆

    PubMed Central

    Saniei, Mansooreh

    2013-01-01

    The paper is based on a large qualitative study of ethics, policy and regulation of human embryonic stem cell (hESC) science in Iran. This case study in five academic research centres used semi-structured interviews to examine in depth the views of stem cell scientists, embryologists and ethics committee members on hESC research policy in this Shia Muslim country. Although Iran's policy approach has been considered 'intermediate', what is described here seems to be a 'more flexible' policy on hESC science. This article describes three arguments to explain why Iran has shaped such a policy. These are: (1) a flexibility of the Shia tradition has allowed for hESC science; (2) permissive policy related to other fields of biomedicine, such as new assisted reproductive technologies, facilitated approval of hESC research; and (3) a lack of public debate of bioscience in Iran influences how its hESC research policy is perceived. Based on the empirical data, this paper then expands and refines the conceptual bioethical basis for the co-production of science, policy, and society in Iran. The notion of co-production implies that scientists, policy-makers, and sometimes other societal actors cooperate in the exchange, production, and application of knowledge to make science policy. PMID:24230960

  13. Human embryonic stem cell science and policy: the case of Iran.

    PubMed

    Saniei, Mansooreh

    2013-12-01

    The paper is based on a large qualitative study of ethics, policy and regulation of human embryonic stem cell (hESC) science in Iran. This case study in five academic research centres used semi-structured interviews to examine in depth the views of stem cell scientists, embryologists and ethics committee members on hESC research policy in this Shia Muslim country. Although Iran's policy approach has been considered 'intermediate', what is described here seems to be a 'more flexible' policy on hESC science. This article describes three arguments to explain why Iran has shaped such a policy. These are: (1) a flexibility of the Shia tradition has allowed for hESC science; (2) permissive policy related to other fields of biomedicine, such as new assisted reproductive technologies, facilitated approval of hESC research; and (3) a lack of public debate of bioscience in Iran influences how its hESC research policy is perceived. Based on the empirical data, this paper then expands and refines the conceptual bioethical basis for the co-production of science, policy, and society in Iran. The notion of co-production implies that scientists, policy-makers, and sometimes other societal actors cooperate in the exchange, production, and application of knowledge to make science policy.

  14. Solar cells. Electron-hole diffusion lengths > 175 μm in solution-grown CH3NH3PbI3 single crystals.

    PubMed

    Dong, Qingfeng; Fang, Yanjun; Shao, Yuchuan; Mulligan, Padhraic; Qiu, Jie; Cao, Lei; Huang, Jinsong

    2015-02-27

    Long, balanced electron and hole diffusion lengths greater than 100 nanometers in the polycrystalline organolead trihalide compound CH3NH3PbI3 are critical for highly efficient perovskite solar cells. We found that the diffusion lengths in CH3NH3PbI3 single crystals grown by a solution-growth method can exceed 175 micrometers under 1 sun (100 mW cm(-2)) illumination and exceed 3 millimeters under weak light for both electrons and holes. The internal quantum efficiencies approach 100% in 3-millimeter-thick single-crystal perovskite solar cells under weak light. These long diffusion lengths result from greater carrier mobility, longer lifetime, and much smaller trap densities in the single crystals than in polycrystalline thin films. The long carrier diffusion lengths enabled the use of CH3NH3PbI3 in radiation sensing and energy harvesting through the gammavoltaic effect, with an efficiency of 3.9% measured with an intense cesium-137 source.

  15. Multi-stacked InAs/GaAs quantum dots grown with different growth modes for quantum dot solar cells

    SciTech Connect

    Kim, Yeongho; Ban, Keun-Yong Honsberg, Christiana B.

    2015-06-01

    We have studied the material properties and device performance of InAs/GaAs quantum dot solar cells (QDSCs) made using three different QD growth modes: Stranski-Krastanov (S-K), quasi-monolayer (QML), and sub-monolayer (SML) growth modes. All QDSCs show an extended external quantum efficiency (EQE) at near infrared wavelengths of 950–1070 nm from the QD absorption. Compared to the S-K and SML QDSCs, the QML QDSC with a higher strain exhibits a poor EQE response in the wavelength region of 300–880 nm due to increased non-radiative recombination. The conversion efficiency of the S-K and SML QDSCs exceeds that of the reference cell (13.4%) without QDs due to an enhanced photocurrent (>16% increase) produced by the silicon doped QD stacks. However, as expected from the EQE of the QML QDSC, the increase of strain-induced crystalline defects greatly degrades the photocurrent and open-circuit voltage, leading to the lowest conversion efficiency (8.9%)

  16. Targeting of the Human Coagulation Factor IX Gene at rDNA Locus of Human Embryonic Stem Cells

    PubMed Central

    Yang, Junlin; Xue, Jinfeng; Hu, Youjin; Feng, Mai; Niu, Wenbin; Yang, Qiurui; Lei, Ming; Xia, Jiahui; Wu, Lingqian; Liang, Desheng

    2012-01-01

    Background Genetic modification is a prerequisite to realizing the full potential of human embryonic stem cells (hESCs) in human genetic research and regenerative medicine. Unfortunately, the random integration methods that have been the primary techniques used keep creating problems, and the primary alternative method, gene targeting, has been effective in manipulating mouse embryonic stem cells (mESCs) but poorly in hESCs. Methodology/Principal Findings Human ribosomal DNA (rDNA) repeats are clustered on the short arm of acrocentric chromosomes. They consist of approximately 400 copies of the 45S pre-RNA (rRNA) gene per haploid. In the present study, we targeted a physiological gene, human coagulation factor IX, into the rDNA locus of hESCs via homologous recombination. The relative gene targeting efficiency (>50%) and homologous recombination frequency (>10−5) were more than 10-fold higher than those of loci targeted in previous reports. Meanwhile, the targeted clones retained both a normal karyotype and the main characteristics of ES cells. The transgene was found to be stably and ectopically expressed in targeted hESCs. Conclusion/Significance This is the first targeting of a human physiological gene at a defined locus on the hESC genome. Our findings indicate that the rDNA locus may serve as an ideal harbor for transgenes in hESCs. PMID:22615895

  17. Effects of Malignant Effusions on the Mitotic Index of L Strain Mouse Cells Grown in Tissue Culture

    PubMed Central

    Hrushovetz, S. B.; Ewaniuk, Meriam H.

    1963-01-01

    By employing a clone of L strain mouse fibroblasts (LE) which does not exhibit cell clumping and lysis (cytolytic antibody reaction), it was possible to screen for the presence of growth-regulating factors in human sera and effusions, exclusive of an antigen-antibody reaction. Under conditions of the test a mitotic index greater than 20% indicated the presence of a growth-promoting factor. A total of 11 pleural effusions was tested. Four of the eight malignant effusions possessed a growth-promoting factor, while none of the three non-malignant effusions or the one sample of human umbilical cord serum possessed such a factor. Overnight storage of the unfiltered effusions at 5° C. resulted in complete loss of the growthpromoting activity. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6 PMID:14052976

  18. Eyes open to stem cells: safety trial may pave the way for cell therapy to treat retinal disease in patients

    PubMed Central

    2011-01-01

    A clinical trial using human embryonic stem cell (hESC) therapy for an inherited retinal degenerative disease is about to commence. The Advanced Cell Technology (ACT) trial will treat patients with Stargardt's macular dystrophy using transplanted retinal pigment epithelium derived from hESCs. Currently, no effective treatment is available for Stargardt's disease so a stem cell-based therapy that can slow progression of this blinding condition could represent a significant breakthrough. While there are some hurdles to clear, the ACT trial is a fine example of translational research that could eventually pave the way for a range of stem cell therapies for the retina and other tissues. PMID:22152341

  19. Effects of chondrogenic and osteogenic regulatory factors on composite constructs grown using human mesenchymal stem cells, silk scaffolds and bioreactors.

    PubMed

    Augst, Alexander; Marolt, Darja; Freed, Lisa E; Vepari, Charu; Meinel, Lorenz; Farley, Michelle; Fajardo, Robert; Patel, Nipun; Gray, Martha; Kaplan, David L; Vunjak-Novakovic, Gordana

    2008-08-01

    Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite. PMID:18230586

  20. Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale.

    PubMed

    Fibi, M R; Bröker, M; Schulz, R; Johannsen, R; Zettlmeissl, G

    1991-08-01

    Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taq-polymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121 degrees C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10(3) per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120 degrees C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes.

  1. Inhibition of vimentin or B1 integrin reverts morphology of prostate tumor cells grown in laminin-rich extracellular matrix gels and reduces tumor growth in vivo

    SciTech Connect

    Zhang, Xueping; Fournier, Marcia V; Ware, Joy L; Bissell, Mina J; Yacoub, Adly; Zehner, Zendra E

    2008-06-12

    Prostate epithelial cells grown embedded in laminin-rich extracellular matrix (lrECM) undergo morphologic changes that closely resemble their architecture in vivo. In this study, growth characteristics of three human prostate epithelial sublines derived from the same cellular lineage, but displaying different tumorigenic and metastatic properties in vivo, were assessed in three-dimensional lrECM gels. M12, a highly tumorigenic and metastatic subline, was derived from the immortalized, prostate epithelial P69 cell line by selection in athymic, nude mice and found to contain a deletion of 19p-q13.1. The stable reintroduction of an intact human chromosome 19 into M12 resulted in a poorly tumorigenic subline, designated F6. When embedded in lrECM gels, the parental, nontumorigenic P69 line produced acini with clearly defined lumena. Immunostaining with antibodies to {beta}-catenin, E-cadherin, or {alpha}6 and {beta}1 integrins showed polarization typical of glandular epithelium. In contrast, the metastatic M12 subline produced highly disorganized cells with no evidence of polarization. The F6 subline reverted to acini-like structures exhibiting basal polarity marked with integrins. Reducing either vimentin levels via small interfering RNA interference or the expression of {alpha}6 and {beta}1 integrins by the addition of blocking antibodies, reorganized the M12 subline into forming polarized acini. The loss of vimentin significantly reduced M12-Vim tumor growth when assessed by s.c. injection in athymic mice. Thus, tumorigenicity in vivo correlated with disorganized growth in three-dimensional lrECM gels. These studies suggest that the levels of vimentin and {beta}1 integrin play a key role in the homeostasis of the normal acinus in prostate and that their dysregulation may lead to tumorigenesis. [Mol Cancer Ther 2009;8(3):499-508].

  2. Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells.

    PubMed

    Ishii, Takamichi; Fukumitsu, Ken; Yasuchika, Kentaro; Adachi, Keiko; Kawase, Eihachiro; Suemori, Hirofumi; Nakatsuji, Norio; Ikai, Iwao; Uemoto, Shinji

    2008-08-01

    Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.

  3. Metabolic Regulation of “Ca. Methylacidiphilum Fumariolicum” SolV Cells Grown Under Different Nitrogen and Oxygen Limitations

    PubMed Central

    Khadem, Ahmad F.; Pol, Arjan; Wieczorek, Adam S.; Jetten, Mike S. M.; Op den Camp, Huub J. M.

    2012-01-01

    Aerobic methanotrophic bacteria can use methane as their sole energy source. The discovery of “Ca. Methylacidiphilum fumariolicum” strain SolV and other verrucomicrobial methanotrophs has revealed that the ability of bacteria to oxidize CH4 is much more diverse than has previously been assumed in terms of ecology, phylogeny, and physiology. A remarkable characteristic of the methane-oxidizing Verrucomicrobia is their extremely acidophilic phenotype, growing even below pH 1. In this study we used RNA-Seq to analyze the metabolic regulation of “Ca. M. fumariolicum” SolV cells growing at μmax in batch culture or under nitrogen fixing or oxygen limited conditions in chemostats, all at pH 2. The analysis showed that two of the three pmoCAB operons each encoding particulate methane monoxygenases were differentially expressed, probably regulated by the available oxygen. The hydrogen produced during N2 fixation is apparently recycled as demonstrated by the upregulation of the genes encoding a Ni/Fe-dependent hydrogenase. These hydrogenase genes were also upregulated under low oxygen conditions. Handling of nitrosative stress was shown by the expression of the nitric oxide reductase encoding genes norB and norC under all conditions tested, the upregulation of nitrite reductase nirK under oxygen limitation and of hydroxylamine oxidoreductase hao in the presence of ammonium. Unraveling the gene regulation of carbon and nitrogen metabolism helps to understand the underlying physiological adaptations of strain SolV in view of the harsh conditions of its natural ecosystem. PMID:22848206

  4. Laser-induced fusion of human embryonic stem cells with optical tweezers

    SciTech Connect

    Chen Shuxun; Wang Xiaolin; Sun Dong; Cheng Jinping; Han Cheng, Shuk; Kong, Chi-Wing; Li, Ronald A.

    2013-07-15

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  5. Laser-induced fusion of human embryonic stem cells with optical tweezers

    NASA Astrophysics Data System (ADS)

    Chen, Shuxun; Cheng, Jinping; Kong, Chi-Wing; Wang, Xiaolin; Han Cheng, Shuk; Li, Ronald A.; Sun, Dong

    2013-07-01

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  6. Histone modifications and p53 binding poise the p21 promoter for activation in human embryonic stem cells

    PubMed Central

    Itahana, Yoko; Zhang, Jinqiu; Göke, Jonathan; Vardy, Leah A.; Han, Rachel; Iwamoto, Kozue; Cukuroglu, Engin; Robson, Paul; Pouladi, Mahmoud A.; Colman, Alan; Itahana, Koji

    2016-01-01

    The high proliferation rate of embryonic stem cells (ESCs) is thought to arise partly from very low expression of p21. However, how p21 is suppressed in ESCs has been unclear. We found that p53 binds to the p21 promoter in human ESCs (hESCs) as efficiently as in differentiated human mesenchymal stem cells, however it does not promote p21 transcription in hESCs. We observed an enrichment for both the repressive histone H3K27me3 and activating histone H3K4me3 chromatin marks at the p21 locus in hESCs, suggesting it is a suppressed, bivalent domain which overrides activation by p53. Reducing H3K27me3 methylation in hESCs rescued p21 expression, and ectopic expression of p21 in hESCs triggered their differentiation. Further, we uncovered a subset of bivalent promoters bound by p53 in hESCs that are similarly induced upon differentiation in a p53-dependent manner, whereas p53 promotes the transcription of other target genes which do not show an enrichment of H3K27me3 in ESCs. Our studies reveal a unique epigenetic strategy used by ESCs to poise undesired p53 target genes, thus balancing the maintenance of pluripotency in the undifferentiated state with a robust response to differentiation signals, while utilizing p53 activity to maintain genomic stability and homeostasis in ESCs. PMID:27346849

  7. Histone modifications and p53 binding poise the p21 promoter for activation in human embryonic stem cells.

    PubMed

    Itahana, Yoko; Zhang, Jinqiu; Göke, Jonathan; Vardy, Leah A; Han, Rachel; Iwamoto, Kozue; Cukuroglu, Engin; Robson, Paul; Pouladi, Mahmoud A; Colman, Alan; Itahana, Koji

    2016-06-27

    The high proliferation rate of embryonic stem cells (ESCs) is thought to arise partly from very low expression of p21. However, how p21 is suppressed in ESCs has been unclear. We found that p53 binds to the p21 promoter in human ESCs (hESCs) as efficiently as in differentiated human mesenchymal stem cells, however it does not promote p21 transcription in hESCs. We observed an enrichment for both the repressive histone H3K27me3 and activating histone H3K4me3 chromatin marks at the p21 locus in hESCs, suggesting it is a suppressed, bivalent domain which overrides activation by p53. Reducing H3K27me3 methylation in hESCs rescued p21 expression, and ectopic expression of p21 in hESCs triggered their differentiation. Further, we uncovered a subset of bivalent promoters bound by p53 in hESCs that are similarly induced upon differentiation in a p53-dependent manner, whereas p53 promotes the transcription of other target genes which do not show an enrichment of H3K27me3 in ESCs. Our studies reveal a unique epigenetic strategy used by ESCs to poise undesired p53 target genes, thus balancing the maintenance of pluripotency in the undifferentiated state with a robust response to differentiation signals, while utilizing p53 activity to maintain genomic stability and homeostasis in ESCs.

  8. Emotion in political discourse: contrasting approaches to stem cell governance in the USA, UK, Israel and Germany.

    PubMed

    Gottweis, Herbert; Prainsack, Barbara

    2006-11-01

    In August 2004, Stojkovic and Murdoch from the University of Newcastle upon Tyne, UK, were granted the UK's first license to create human embryonic stem cells (hESCs) using cell nuclear replacement. While this news made headlines around the globe, a spokesman for the German Ministry of Research warned scientists in his country of the illegality of advising their English colleagues on hESC research. Meanwhile, US Members of Congress had asked President Bush to revoke his decision to limit federal funding to research on a limited number of hESC lines created before 9 August, 2001 (a decision that he confirmed in July 2006, while nonfederally funded research on hESC continues to be unrestricted). In Israel, where hESC research is legal and has never been a contested political issue, a bioethicist argued that, in light of the potential to alleviate human suffering, "banning research is against human dignity". How can such striking differences in the regulation of hESC research be explained? PMID:17465763

  9. Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State

    PubMed Central

    Pells, Steve; Koutsouraki, Eirini; Morfopoulou, Sofia; Valencia-Cadavid, Sara; Tomlinson, Simon R.; Kalathur, Ravi; Futschik, Matthias E.; De Sousa, Paul A.

    2015-01-01

    Human embryonic stem cells (hESCs) undergo epigenetic changes in vitro which may compromise function, so an epigenetic pluripotency “signature” would be invaluable for line validation. We assessed Cytosine-phosphate-Guanine Island (CGI) methylation in hESCs by genomic DNA hybridisation to a CGI array, and saw substantial variation in CGI methylation between lines. Comparison of hESC CGI methylation profiles to corresponding somatic tissue data and hESC mRNA expression profiles identified a conserved hESC-specific methylation pattern associated with expressed genes. Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively. Knockdown of candidate transcriptional regulators (HMGA1, GLIS2, PFDN5) induced differentiation in hESCs, whereas ectopic expression in fibroblasts modulated iPSC colony formation. Chromatin immunoprecipitation confirmed interaction between the candidates and the core pluripotency transcription factor network. We thus identify novel pluripotency genes on the basis of a conserved and distinct epigenetic configuration in human stem cells. PMID:26151932

  10. Histone modifications and p53 binding poise the p21 promoter for activation in human embryonic stem cells.

    PubMed

    Itahana, Yoko; Zhang, Jinqiu; Göke, Jonathan; Vardy, Leah A; Han, Rachel; Iwamoto, Kozue; Cukuroglu, Engin; Robson, Paul; Pouladi, Mahmoud A; Colman, Alan; Itahana, Koji

    2016-01-01

    The high proliferation rate of embryonic stem cells (ESCs) is thought to arise partly from very low expression of p21. However, how p21 is suppressed in ESCs has been unclear. We found that p53 binds to the p21 promoter in human ESCs (hESCs) as efficiently as in differentiated human mesenchymal stem cells, however it does not promote p21 transcription in hESCs. We observed an enrichment for both the repressive histone H3K27me3 and activating histone H3K4me3 chromatin marks at the p21 locus in hESCs, suggesting it is a suppressed, bivalent domain which overrides activation by p53. Reducing H3K27me3 methylation in hESCs rescued p21 expression, and ectopic expression of p21 in hESCs triggered their differentiation. Further, we uncovered a subset of bivalent promoters bound by p53 in hESCs that are similarly induced upon differentiation in a p53-dependent manner, whereas p53 promotes the transcription of other target genes which do not show an enrichment of H3K27me3 in ESCs. Our studies reveal a unique epigenetic strategy used by ESCs to poise undesired p53 target genes, thus balancing the maintenance of pluripotency in the undifferentiated state with a robust response to differentiation signals, while utilizing p53 activity to maintain genomic stability and homeostasis in ESCs. PMID:27346849

  11. Nickel nanocrystals grown on sparse hierarchical CuS microflowers as high-performance counter electrodes for dye-sensitized solar cells

    NASA Astrophysics Data System (ADS)

    Shi, Zhaoliang; Zhou, Wei; Ma, Yiran

    2016-07-01

    Three kinds of hierarchical CuS microflowers composed of thin nanosheets have been synthesized by a simple wet chemical method. It is shown that the CuS microflowers provide suitable substrates to grow nickel nanocrystals. The prepared Ni@CuS hybrids combined with conductive glass (FTO) have been used as counter electrodes for dye-sensitized solar cells (DSSCs). The electrode made of the active material of Ni@CuS microflowers with sparsest petals show an optimal photoelectric conversion efficiency of 4.89%, better than those made of single component of Ni (3.39%) or CuS (1.65%), and other two Ni@CuS composites. The improved performances could be ascribed to the synergetic effect of the catalytic effect towards I3‑/I‑ from sparse CuS hierarchical structure and uniformly grown Ni nanocrystals. Besides, the introduced Ni nanocrystals could increase the conductivity of the hybrid and facilitate the transport of electrons. The hybrid Ni@CuS composites serving as counter electrodes have much enhanced electrochemical properties, which provide a feasible route to develop high-active non-noble hybrid counter electrode materials.

  12. Uptake of cadmium by rice grown on contaminated soils and its bioavailability/toxicity in human cell lines (Caco-2/HL-7702).

    PubMed

    Aziz, Rukhsanda; Rafiq, Muhammad Tariq; Li, Tingqiang; Liu, Di; He, Zhenli; Stoffella, P J; Sun, Kewang; Xiaoe, Yang

    2015-04-01

    Cadmium (Cd) enters the food chain from polluted soils via contaminated cereals and vegetables; therefore, an understanding of Cd bioaccessibility, bioavailability, and toxicity in humans through rice grain is needed. This study assessed the Cd bioaccessibility, bioavailability, and toxicity to humans from rice grown on Cd-contaminated soils using an in vitro digestion method combined with a Caco-2/HL-7702 cell model. Cadmium bioaccessibility (18.45-30.41%) and bioavailability (4.04-8.62%) were found to be significantly higher in yellow soil (YS) rice than calcareous soil (CS) rice with the corresponding values of 6.89-11.43 and 1.77-2.25%, respectively. Toxicity assays showed an initial toxicity in YS rice at 6 mg kg(-1) Cd, whereas CS rice did not show any significant change due to low Cd concentrations. The acidic soils of Cd-contaminated areas can contribute to a higher dietary intake of Cd. Therefore, it is imperative to monitor Cd concentration in rice to minimize human health risk.

  13. Graphene grown on stainless steel as a high-performance and ecofriendly anti-corrosion coating for polymer electrolyte membrane fuel cell bipolar plates

    NASA Astrophysics Data System (ADS)

    Pu, Nen-Wen; Shi, Gia-Nan; Liu, Yih-Ming; Sun, Xueliang; Chang, Jeng-Kuei; Sun, Chia-Liang; Ger, Ming-Der; Chen, Chun-Yu; Wang, Po-Chiang; Peng, You-Yu; Wu, Chia-Hung; Lawes, Stephen

    2015-05-01

    In this study, the growth of graphene by chemical vapor deposition (CVD) on SUS304 stainless steel and on a catalyzing Ni/SUS304 double-layered structure was investigated. The results indicated that a thin and multilayered graphene film can be continuously grown across the metal grain boundaries of the Ni/SUS304 stainless steel and significantly enhance its corrosion resistance. A 3.5 wt% saline polarization test demonstrated that the corrosion currents in graphene-covered SUS304 were improved fivefold relative to the corrosion currents in non-graphene-covered SUS304. In addition to enhancing the corrosion resistance of stainless steel, a graphene coating also ameliorates another shortcoming of stainless steel in a corrosive environment: the formation of a passive oxidation layer on the stainless steel surface that decreases conductivity. After a corrosion test, the graphene-covered stainless steel continued to exhibit not only an excellent low interfacial contact resistance (ICR) of 36 mΩ cm2 but also outstanding drainage characteristics. The above results suggest that an extremely thin, lightweight protective coating of graphene on stainless steel can act as the next-generation bipolar plates of fuel cells.

  14. A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells

    SciTech Connect

    Liu Yanxia; Song Zhihua; Zhao Yang; Qin Han; Cai Jun; Zhang Hong; Yu Tianxin; Jiang Siming; Wang Guangwen; Ding Mingxiao; Deng Hongkui . E-mail: hongkui_deng@pku.edu.cn

    2006-07-21

    Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs.

  15. Constraining the Pluripotent Fate of Human Embryonic Stem Cells for Tissue Engineering and Cell Therapy – The Turning Point of Cell-Based Regenerative Medicine

    PubMed Central

    Parsons, Xuejun H.

    2014-01-01

    To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for regenerating the damaged or lost CNS structure and circuitry in a wide range of neurological disorders. Similarly, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart. Given the limited capacity of the CNS and heart for self-repair, there is a large unmet healthcare need to develop stem cell therapies to provide optimal regeneration and reconstruction treatment options to restore normal tissues and function. Derivation of human embryonic stem cells (hESCs) provides a powerful in vitro model system to investigate molecular controls in human embryogenesis as well as an unlimited source to generate the diversity of human somatic cell types for regenerative medicine. However, realizing the developmental and therapeutic potential of hESC derivatives has been hindered by the inefficiency and instability of generating clinically-relevant functional cells from pluripotent cells through conventional uncontrollable and incomplete multi-lineage differentiation. Recent advances and breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing defined culture systems for de novo derivation and maintenance of clinical-grade pluripotent hESCs and lineage-specific differentiation of pluripotent hESCs by small molecule induction. Retinoic acid was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors and neurons of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting

  16. Effect of antibiotics against Mycoplasma sp. on human embryonic stem cells undifferentiated status, pluripotency, cell viability and growth.

    PubMed

    Romorini, Leonardo; Riva, Diego Ariel; Blüguermann, Carolina; Videla Richardson, Guillermo Agustin; Scassa, Maria Elida; Sevlever, Gustavo Emilio; Miriuka, Santiago Gabriel

    2013-01-01

    Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of Plasmocin(TM) and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days Plasmocin(TM) 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with Plasmocin(TM) 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that Plasmocin(TM) and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.

  17. Epigenetic reprogramming of human embryonic stem cells into skeletal muscle cells and generation of contractile myospheres.

    PubMed

    Albini, Sonia; Coutinho, Paula; Malecova, Barbora; Giordani, Lorenzo; Savchenko, Alex; Forcales, Sonia Vanina; Puri, Pier Lorenzo

    2013-03-28

    Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for "disease in a dish" models of muscular physiology and dysfunction.

  18. Video of Tissue Grown in Space in NASA Bioreactor

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Principal investigator Leland Chung grew prostate cancer and bone stromal cells aboard the Space Shuttle Columbia during the STS-107 mission. Although the experiment samples were lost along with the ill-fated spacecraft and crew, he did obtain downlinked video of the experiment that indicates the enormous potential of growing tissues in microgravity. Cells grown aboard Columbia had grown far larger tissue aggregates at day 5 than did the cells grown in a NASA bioreactor on the ground.

  19. Temporal impact of substrate mechanics on differentiation of human embryonic stem cells to cardiomyocytes.

    PubMed

    Hazeltine, Laurie B; Badur, Mehmet G; Lian, Xiaojun; Das, Amritava; Han, Wenqing; Palecek, Sean P

    2014-02-01

    A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact the efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces, which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes.

  20. Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells

    PubMed Central

    Mandegar, Mohammad A.; Moralli, Daniela; Khoja, Suhail; Cowley, Sally; Chan, David Y.L.; Yusuf, Mohammed; Mukherjee, Sayandip; Blundell, Michael P.; Volpi, Emanuela V.; Thrasher, Adrian J.; James, William; Monaco, Zoia L.

    2011-01-01

    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. PMID:21593218

  1. Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells.

    PubMed

    Mandegar, Mohammad A; Moralli, Daniela; Khoja, Suhail; Cowley, Sally; Chan, David Y L; Yusuf, Mohammed; Mukherjee, Sayandip; Blundell, Michael P; Volpi, Emanuela V; Thrasher, Adrian J; James, William; Monaco, Zoia L

    2011-08-01

    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. PMID:21593218

  2. [Development of human embryonic stem cell platforms for human health-safety evaluation].

    PubMed

    Yu, Guang-yan; Cao, Tong; Zou, Xiao-hui; Zhang, Xue-hui; Fu, Xin; Peng, Shuang-qing; Deng, Xu-liang; Li, Sheng-lin; Liu, He; Xiao, Ran; Ouyang, Hong-wei; Peng, Hui; Chen, Xiao; Zhao, Zeng-ming; Wang, Xiao-ying; Fang, Hai-qin; Lu, Lu; Ren, Yu-lan; Xu, Ming-ming

    2016-02-18

    The human embryonic stem cells (hESCs) serve as a self-renewable, genetically-healthy, pluripotent and single source of all body cells, tissues and organs. Therefore, it is considered as the good standard for all human stem cells by US, Europe and international authorities. In this study, the standard and healthy human mesenchymal progenitors, ligament tissues, cardiomyocytes, keratinocytes, primary neurons, fibroblasts, and salivary serous cells were differentiated from hESCs. The human cellular health-safety of NaF, retinoic acid, 5-fluorouracil, dexamethasone, penicillin G, adriamycin, lead acetate PbAc, bisphenol A-biglycidyl methacrylate (Bis-GMA) were evaluated selectively on the standardized platforms of hESCs, hESCs-derived cardiomyocytes, keratinocytes, primary neurons, and fibroblasts. The evaluations were compared with those on the currently most adopted cellular platforms. Particularly, the sensitivity difference of PM2.5 toxicity on standardized and healthy hESCs derived fibroblasts, currently adopted immortalized human bronchial epithelial cells Beas-2B and human umbilical vein endothelial cells (HUVECs) were evaluated. The RESULTS showed that the standardized hESCs cellular platforms provided more sensitivity and accuracy for human cellular health-safety evaluation. PMID:26885900

  3. Effect of passivation layer grown by atomic layer deposition and sputtering processes on Si quantum dot superlattice to generate high photocurrent for high-efficiency solar cells

    NASA Astrophysics Data System (ADS)

    Maksudur Rahman, Mohammad; Higo, Akio; Sekhar, Halubai; Erman Syazwan, Mohd; Hoshi, Yusuke; Usami, Noritaka; Samukawa, Seiji

    2016-03-01

    The effect of passivation films on a Si quantum dot superlattice (QDSL) was investigated to generate high photocurrent in solar-cell applications. Three types of passivation films, sputter-grown amorphous silicon carbide (a-SiC), hydrogenated a-SiC (a-SiC:H), and atomic-layer-deposited aluminum oxide (ALD-Al2O3), were used to passivate the Si QDSLs containing a stack of four 4 nm Si nanodisks (NDs) and 2 nm silicon carbide (SiC) films fabricated by neutral beam etching (NBE). Because of the high surface-to-volume ratio typically present in quantum Si-NDs formed in the top-down NBE process, there is a tendency to form larger surface dangling bonds on untreated Si-ND surfaces as well as to have short distance (<10 nm) between high-aspect-ratio nanopillars of stacked 4 nm Si-NDs/2 nm SiC films, which conventionally sputter SiC films cannot uniformly cover. Therefore, we optimized the passivation techniques with an ALD-Al2O3 film. Scanning electron microscopy (SEM) analysis helped to explain the surface morphology before and after the passivation of the QDSLs. After the completion of the passivation process, the quality of the top surface films of the QDSLs was analyzed from the surface roughness by atomic force microscopy (AFM) analysis, which revealed that ALD-Al2O3 passivated films had the smallest roughness (RMS) of 1.09 nm with respect to sputter-grown a-SiC (RMS: 1.75 nm) and a-SiC:H (RMS: 1.54 nm) films. Conductive atomic force microscopy (CAFM) revealed that ALD-Al2O3 passivation decreased the surface-leakage current as a result of proper passivation of side-wall surface defects in the QDSLs. The carrier transport characteristics were extracted from the QDSLs using the photovoltaic (PV) properties of p++/i/n+ solar cells, where the QDSLs consisted of different passivation layers acting as intermediate layers (i-layers) between the high-doping-density p++ Si (1 × 1020 cm-3) and n+ Si (1 × 1019 cm-3) substrates. High-doping-density p++ Si acted as a hole

  4. Publishing SNP genotypes of human embryonic stem cell lines: policy statement of the International Stem Cell Forum Ethics Working Party.

    PubMed

    Knoppers, Bartha M; Isasi, Rosario; Benvenisty, Nissim; Kim, Ock-Joo; Lomax, Geoffrey; Morris, Clive; Murray, Thomas H; Lee, Eng Hin; Perry, Margery; Richardson, Genevra; Sipp, Douglas; Tanner, Klaus; Wahlström, Jan; de Wert, Guido; Zeng, Fanyi

    2011-09-01

    Novel methods and associated tools permitting individual identification in publicly accessible SNP databases have become a debatable issue. There is growing concern that current technical and ethical safeguards to protect the identities of donors could be insufficient. In the context of human embryonic stem cell research, there are no studies focusing on the probability that an hESC line donor could be identified by analyzing published SNP profiles and associated genotypic and phenotypic information. We present the International Stem Cell Forum (ISCF) Ethics Working Party's Policy Statement on "Publishing SNP Genotypes of Human Embryonic Stem Cell Lines (hESC)". The Statement prospectively addresses issues surrounding the publication of genotypic data and associated annotations of hESC lines in open access databases. It proposes a balanced approach between the goals of open science and data sharing with the respect for fundamental bioethical principles (autonomy, privacy, beneficence, justice and research merit and integrity).

  5. Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

    PubMed

    Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2015-02-25

    Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process.

  6. YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

    PubMed

    Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

    2012-03-01

    The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

  7. An ESRG-interacting protein, COXII, is involved in pro-apoptosis of human embryonic stem cells.

    PubMed

    Shi, Jia; Ren, Caiping; Liu, Hui; Wang, Lei; Zhu, Bin; Huang, Wei; Liu, Weidong; Liu, Jie; Liu, Yanyu; Xia, Xiaomeng; Xu, Rong; Jiang, Xingjun

    2015-05-01

    Human embryonic stem cells(hESC) posses very promising application perspective in clinical transplant therapies for their characteristics of self-renewal and pluripotency. So efforts focusing on the mechanisms of the two characteristics are extremely important. ESRG, first identified by our group, is a candidate stemness gene of hESC for its much higher expression level in hESC comparing to that in 7-day embryoid bodies(EBs). Here, the proteins interacted with ESRG and its functions in hESC were explored. Yeast two-hybrid (Y2H) screening system was adopted to explore the interacting proteins of ESRG. Then Co-IP was performed to confirm the interactions between candidate proteins and ESRG. At last, the functions of validated interacting protein were explored by RNA interference(RNAi) and Western blot(WB). There were no autonomous activation and toxicity in the Y2H system, which verified its availability. Four candidate proteins, AAMP, DDT, GNB2L1 and COXII, were discovered, and the interaction between ESRG and COXII was ultimately confirmed. The expression of COXII in hESC was suppressed by siRNA, and the inhibited mitochondrial apoptosis was observed in hESC with downregulated COXII expression. Our work first validated the interaction between ESRG and COXII, and demonstrated that COXII serves as a pro-apoptotic protein in hESC. The results implied that ESRG may play an important role in regulating the apoptosis of hESC by interacting with COXII, and thus contribute a lot to the maintenance of hESC characteristics. PMID:25748575

  8. Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

    PubMed Central

    Wang, Linlin; Schulz, Thomas C.; Sherrer, Eric S.; Dauphin, Derek S.; Shin, Soojung; Nelson, Angelique M.; Ware, Carol B.; Zhan, Mei; Song, Chao-Zhong; Chen, Xiaoji; Brimble, Sandii N.; McLean, Amanda; Galeano, Maria J.; Uhl, Elizabeth W.; D'Amour, Kevin A.; Chesnut, Jonathan D.; Rao, Mahendra S.

    2007-01-01

    Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs. PMID:17761519

  9. Derivation of Xeno-Free and GMP-Grade Human Embryonic Stem Cells – Platforms for Future Clinical Applications

    PubMed Central

    Aizenman, Einat; Kirshberg, Sophie; Ilouz, Nili; Gil, Yaniv; Berman-Zaken, Yael; Perlman, Temima Schnitzer; Geva, Nitshia; Levy, Ora; Arbell, Daniel; Simon, Alex; Ben-Meir, Assaf; Shufaro, Yoel; Laufer, Neri; Reubinoff, Benjamin E.

    2012-01-01

    Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs. PMID:22745653

  10. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

    PubMed

    Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

    2015-01-01

    We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve. PMID:26473610

  11. Human Dendritic Cells Derived From Embryonic Stem Cells Stably Modified With CD1d Efficiently Stimulate Antitumor Invariant Natural Killer T Cell Response

    PubMed Central

    2014-01-01

    Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However, the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study, we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette, we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly, more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification, and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional, as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore, hESCs stably modified with the CD1d gene may serve as a convenient, unlimited, and competent DC source for iNKT cell-based cancer immunotherapy. PMID:24292792

  12. Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers

    PubMed Central

    Fujitani, Naoki; Furukawa, Jun-ichi; Araki, Kayo; Fujioka, Tsuyoshi; Takegawa, Yasuhiro; Piao, Jinhua; Nishioka, Taiki; Tamura, Tomohiro; Nikaido, Toshio; Ito, Makoto; Nakamura, Yukio; Shinohara, Yasuro

    2013-01-01

    Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry–based structurally intensive analyses, both qualitatively and quantitatively, of cellular N- and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell–specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1–60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs. PMID:23345451

  13. The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

    PubMed

    Sperber, Henrik; Mathieu, Julie; Wang, Yuliang; Ferreccio, Amy; Hesson, Jennifer; Xu, Zhuojin; Fischer, Karin A; Devi, Arikketh; Detraux, Damien; Gu, Haiwei; Battle, Stephanie L; Showalter, Megan; Valensisi, Cristina; Bielas, Jason H; Ericson, Nolan G; Margaretha, Lilyana; Robitaille, Aaron M; Margineantu, Daciana; Fiehn, Oliver; Hockenbery, David; Blau, C Anthony; Raftery, Daniel; Margolin, Adam A; Hawkins, R David; Moon, Randall T; Ware, Carol B; Ruohola-Baker, Hannele

    2015-12-01

    For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development. PMID:26571212

  14. Optical and electrical characterization of CdS-Glycine thin films with ammonia free buffer grown at different temperatures for solar cells applications

    NASA Astrophysics Data System (ADS)

    Berman-Mendoza, D.; Quiñones-Urías, D.; Ferra-González, S.; Vera-Marquina, A.; Rojas-Hernández, A.; Gómez Fuentes, R.; García-Juárez, A.; Leal-Cruz, A. L.; Ramos-Carrasco, A.

    2013-11-01

    In this work we report the fabrication and electro-optical characterization of CdS thin films using glycine as complexing agent with ammonia and ammonia free buffer by the Chemical Bath Deposition (CBD) method. The CdS thin films were grown at different temperatures of 50, 60, 70 and 80 °C in a thermal water bath. The morphology of these films was determined using atomic force microscopy; the resultant films were homogeneous, well adhered to the substrate, and specularly reflecting with a varying color depending on the deposition temperature. Transmittance and reflectance measurements of thermally treated CdS films were carried to study the effect of the ammonia buffer on its optical properties and bandgap. The crystallinity of the CdS thin films was determined by means of X Ray diffraction measurements. Therefore, for this study, an ammonia-free complexing agent has been taken for the deposition of CdS. Among different methods, which are being used for the preparation of CdS films, Chemical Bath Deposition (CBD) is the most attractive due to its low cost, easy to handle and large possibilities regarding doping and deposition on various substrates. In particular it can be used to easily obtain field effect devices by depositing CdS thin films over a SiO2/Si substrate. Heterostructures with interesting physical properties can be imagined, realized and tested in this way.. Structures CdS/PbS also were realized and have shown good solar cell characteristics.

  15. Distinct differentiation characteristics of individual human embryonic stem cell lines

    PubMed Central

    Mikkola, Milla; Olsson, Cia; Palgi, Jaan; Ustinov, Jarkko; Palomaki, Tiina; Horelli-Kuitunen, Nina; Knuutila, Sakari; Lundin, Karolina; Otonkoski, Timo; Tuuri, Timo

    2006-01-01

    Background Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. Results The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. Conclusion hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state. PMID:16895598

  16. Stem cell research ethics: consensus statement on emerging issues.

    PubMed

    Caulfield, Timothy; Ogbogu, Ubaka; Nelson, Erin; Einsiedel, Edna; Knoppers, Bartha; McDonald, Michael; Brunger, Fern; Downey, Robin; Fernando, Kanchana; Galipeau, Jacques; Geransar, Rose; Griener, Glenn; Grenier, Glenn; Hyun, Insoo; Isasi, Rosario; Kardel, Melanie; Knowles, Lori; Kucic, Terrence; Lotjonen, Salla; Lyall, Drew; Magnus, David; Mathews, Debra J H; Nisbet, Matthew; Nisker, Jeffrey; Pare, Guillaume; Pattinson, Shaun; Pullman, Daryl; Rudnicki, Michael; Williams-Jones, Bryn; Zimmerman, Susan

    2007-10-01

    This article is a consensus statement by an international interdisciplinary group of academic experts and Canadian policy-makers on emerging ethical, legal and social issues in human embryonic stem cells (hESC) research in Canada. The process of researching consensus included consultations with key stakeholders in hESC research (regulations, stem cell researchers, and research ethics experts), preparation and distribution of background papers, and an international workshop held in Montreal in February 2007 to discuss the papers and debate recommendations. The recommendations provided in the consensus statement focus on issues of immediate relevance to Canadian policy-makers, including informed consent to hESC research, the use of fresh embryos in research, management of conflicts of interest, and the relevance of public opinion research to policy-making.

  17. Catalase Activity of Psychrophilic Bacteria Grown at 2 and 30 C1

    PubMed Central

    Frank, Hilmer A.; Ishibashi, Sandra T.; Reid, Ann; Ito, June S.

    1963-01-01

    Catalase activity was measured in resting-cell suspensions of psychrophilic bacteria grown at 2 and at 30 C. Enzyme activity decreased in both cell-suspension types as harvest age increased. At comparable physiological age, cells grown at 2 C had more catalase than cells grown at 30 C. PMID:13959237

  18. A human embryonic stem cell line adapted for high throughput screening.

    PubMed

    Caron, Nicolas J; Gage, Blair K; O'Connor, Michael D; Eaves, Connie J; Kieffer, Timothy J; Piret, James M

    2013-10-01

    Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However, optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs, including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20, they remain highly useful. In particular, CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus, 10(8) CA1S cells can be generated within one week from 10(6) cells to seed 10(6) wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm, a finding that was confirmed with H1 hESCs, although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines, these cells should ease the development of optimized hESC growth and differentiation protocols. In particular, they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses. PMID:23613129

  19. Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro

    PubMed Central

    Hanson, Charles; Hardarson, Thorir; Ellerström, Catharina; Nordberg, Markus; Caisander, Gunilla; Rao, Mahendra; Hyllner, Johan; Stenevi, Ulf

    2013-01-01

    Purpose The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial-like cells. Methods Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman’s membrane for up to 9 days, and the culture dynamics documented in a time-lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial-like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6. Results The transplanted cells established and expanded on Bowman’s membrane, forming a 1–4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3 days after transplantation, and after 6 days, the cells were expressing both PAX6 and CK3. Conclusion This shows that it is possible to transplant cells originating from hESCs onto Bowman’s membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial-like cells in vitro. PMID:22280565

  20. FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells

    SciTech Connect

    Sui, Lina; Mfopou, Josue K.; Geens, Mieke; Sermon, Karen; Bouwens, Luc

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Deep study the FGF signaling role during DE specification in the context of hESCs. Black-Right-Pointing-Pointer DE differentiation from hESCs has an early dependence on FGF signaling. Black-Right-Pointing-Pointer A serum-free DE protocol is developed based on the findings. Black-Right-Pointing-Pointer The DE cells showed potential to differentiate into pancreatic progenitor cells. -- Abstract: Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study, we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.

  1. Investigation of anodic and chemical oxides grown on p-type InP with applications to surface passivation for n(+)-p solar cell fabrication

    NASA Technical Reports Server (NTRS)

    Faur, Maria; Faur, Mircea; Goradia, Manju; Goradia, Chandra; Jenkins, Phillip; Jayne, Douglas; Weinberg, Irving

    1991-01-01

    Most of the previously reported InP anodic oxides were grown on a n-type InP with applications to fabrication of MISFET structures and were described as a mixture of In2O3 and P2O5 stoichiometric compounds or nonstoichiometric phases which have properties similar to crystalline compounds In(OH)3, InPO4, and In(PO3)3. Details of the compositional change of the anodic oxides grown under different anodization conditions were previously reported. The use of P-rich oxides grown either by anodic or chemical oxidation are investigated for surface passivation of p-type InP and as a protective cap during junction formation by closed-ampoule sulfur diffusion. The investigation is based on but not limited to correlations between PL intensity and X-ray photoelectron spectroscopy (XPS) chemical composition data.

  2. Changes in glycosphingolipid composition during differentiation of human embryonic stem cells to ectodermal or endodermal lineages.

    PubMed

    Liang, Yuh-Jin; Yang, Bei-Chia; Chen, Jin-Mei; Lin, Yu-Hsing; Huang, Chia-Lin; Cheng, Yuan-Yuan; Hsu, Chi-Yen; Khoo, Kay-Hooi; Shen, Chia-Ning; Yu, John

    2011-12-01

    Glycosphingolipids (GSLs) are ubiquitous components of cell membranes that can act as mediators of cell adhesion and signal transduction and can possibly be used as cell type-specific markers. Our previous study indicated that there was a striking switch in the core structures of GSLs during differentiation of human embryonic stem cells (hESCs) into embryoid body (EB), suggesting a close association of GSLs with cell differentiation. In this study, to further clarify if alterations in GSL patterns are correlated with lineage-specific differentiation of hESCs, we analyzed changes in GSLs as hESCs were differentiated into neural progenitors or endodermal cells by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and tandem mass spectrometry (MS/MS) analyses. During hESC differentiation into neural progenitor cells, we found that the core structures of GSLs switched from globo- and lacto- to mostly ganglio-series dominated by GD3. On the other hand, when hESCs were differentiated into endodermal cells, patterns of GSLs totally differed from those observed in EB outgrowth and neural progenitors. The most prominent GSL identified by the MALDI-MS and MS/MS analysis was Gb(4) Ceramide, with no appreciable amount of stage-specific embryonic antigens 3 or 4, or GD3, in endodermal cells. These changes in GSL profiling were accompanied by alterations in the biosynthetic pathways of expressions of key glycosyltransferases. Our findings suggest that changes in GSLs are closely associated with lineage specificity and differentiation of hESCs.

  3. In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice.

    PubMed

    Amps, K J; Jones, M; Baker, D; Moore, H D

    2010-06-01

    The development of efficient and robust methods for the cryopreservation of human embryonic stem cells (hESCs) is important for the production of master and working cell banks for future clinical applications. Such methods must meet requirements of good manufacturing practice (GMP) and maintain genetic stability of the cell line. We investigated the culture of four Shef hESC lines in gas permeable 'culture cassettes' which met GMP compliance. hESCs adhered rapidly to the membrane and colonies displayed good proliferation and expansion. After 5-7 days of culture, hESCs were cryopreserved in situ using 10% dimethyl sulphoxide in foetal calf serum at approximately 1 degrees C/min. This method was compared with a control of standard flask culture and cryopreservation in vials. Post-thaw cassette culture displayed relative proliferation ratios (fold increase above flask/cryovial culture) of 114 (Shef 4), 8.2 (Shef 5), 195 (shef 6) and 17.5 (Shef 7). The proportion of cells expressing pluripotency markers after cryopreservation was consistently greater in cassette culture than for the control with the markers SSEA3 and SSEA4 exhibiting a significant increase (P> or =0.05). The efficiency of cell line culture in cassette was associated with the overall passage number of the cell line. The procedure enables cryopreservation of relatively large quantities of hESCs in situ, whilst returning high yields of viable, undifferentiated stem cells, thereby increasing capacity to scale up with greater efficacy.

  4. The Molecular Karyotype of 25 Clinical-Grade Human Embryonic Stem Cell Lines

    PubMed Central

    Canham, Maurice A.; Van Deusen, Amy; Brison, Daniel R.; De Sousa, Paul A.; Downie, Janet; Devito, Liani; Hewitt, Zoe A.; Ilic, Dusko; Kimber, Susan J.; Moore, Harry D.; Murray, Helen; Kunath, Tilo

    2015-01-01

    The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now becoming a reality. Although the vast majority of hESC lines have been derived for research purposes only, about 50 lines have been established under Good Manufacturing Practice (GMP) conditions. Cell types differentiated from these designated lines may be used as a cell therapy to treat macular degeneration, Parkinson’s, Huntington’s, diabetes, osteoarthritis and other degenerative conditions. It is essential to know the genetic stability of the hESC lines before progressing to clinical trials. We evaluated the molecular karyotype of 25 clinical-grade hESC lines by whole-genome single nucleotide polymorphism (SNP) array analysis. A total of 15 unique copy number variations (CNVs) greater than 100 kb were detected, most of which were found to be naturally occurring in the human population and none were associated with culture adaptation. In addition, three copy-neutral loss of heterozygosity (CN-LOH) regions greater than 1 Mb were observed and all were relatively small and interstitial suggesting they did not arise in culture. The large number of available clinical-grade hESC lines with defined molecular karyotypes provides a substantial starting platform from which the development of pre-clinical and clinical trials in regenerative medicine can be realised. PMID:26607962

  5. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    PubMed

    Hirsch, Matthew L; Fagan, B Matthew; Dumitru, Raluca; Bower, Jacquelyn J; Yadav, Swati; Porteus, Matthew H; Pevny, Larysa H; Samulski, R Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

  6. Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures.

    PubMed

    Sikorski, Darek J; Caron, Nicolas J; VanInsberghe, Michael; Zahn, Hans; Eaves, Connie J; Piret, James M; Hansen, Carl L

    2015-10-01

    Heterogeneity in the clonal outputs of individual human embryonic stem cells (hESCs) confounds analysis of their properties in studies of bulk populations and how to manipulate them for clinical applications. To circumvent this problem we developed a microfluidic device that supports the robust generation of colonies derived from single ESCs. This microfluidic system contains 160 individually addressable chambers equipped for perfusion culture of individual hESCs that could be shown to match the growth rates, marker expression and colony morphologies obtained in conventional cultures. Use of this microfluidic device to analyze the clonal growth kinetics of multiple individual hESCs induced to differentiation revealed variable shifts in the growth rate, area per cell and expression of OCT4 in the progeny of individual hESCs. Interestingly, low OCT4 expression, a slower growth rate and low nuclear to cytoplasmic ratios were found to be correlated responses. This study demonstrates how microfluidic systems can be used to enable large scale live-cell imaging of isolated hESCs exposed to changing culture conditions, to examine how different aspects of their variable responses are correlated. PMID:26059045

  7. Viral Single-Strand DNA Induces p53-Dependent Apoptosis in Human Embryonic Stem Cells

    PubMed Central

    Hirsch, Matthew L.; Fagan, B. Matthew; Dumitru, Raluca; Bower, Jacquelyn J.; Yadav, Swati; Porteus, Matthew H.; Pevny, Larysa H.; Samulski, R. Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication. PMID:22114676

  8. Application of an immunoaffinity-based preconcentration method for mass spectrometric analysis of the O-chain polysaccharide of Aeromonas salmonicida from in vitro- and in vivo-grown cells.

    PubMed

    Wang, Zhan; Liu, Xin; Garduño, Elizabeth; Garduño, Rafael A; Li, Jianjun; Altman, Eleonora

    2009-06-01

    In this study, application of magnetic beads (Dynabeads) coated with Aeromonas salmonicida lipopolysaccharide-specific polyclonal antisera to MS-based characterization of bacterial lipopolysaccharides has been evaluated. The results showed that the affinity-based preconcentration strategy resulted in at least a 100-fold increase in the detection of sensitivity, affording direct capillary electrophoresis (CE)-MS analysis of A. salmonicida lipopolysaccharide O-chain polysaccharide from in vitro-cultured cells. Subsequent CE-MS analysis of in vivo-grown cells of A. salmonicida confirmed significant changes in the structure of the lipopolysaccharide O-chain polysaccharide as a result of in vivo cultivation. PMID:19456871

  9. Growth and characterization of Czochralski-grown n and p-type GaAs for space solar cell substrates. Final Report, 29 May 1981-28 May 1982

    SciTech Connect

    Chen, R.T.

    1983-06-01

    Progress in LEC (liquid encapsulated Czochralski) crystal growth techniques for producing high-quality, 3-inch-diameter, n- and p-type GaAs crystals suitable for solar cell applications is described. The LEC crystals with low dislocation densities and background impurities, high electrical mobilities, good dopant uniformity, and long diffusion lengths were reproducibly grown through control of the material synthesis, growth and doping conditions. The capability for producing these large-area, high-quality substrates should positively impact the manufacturability of highly efficiency, low cost, radiation-hard GaAs solar cells.

  10. Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells.

    PubMed

    Enver, Tariq; Soneji, Shamit; Joshi, Chirag; Brown, John; Iborra, Francisco; Orntoft, Torben; Thykjaer, Thomas; Maltby, Edna; Smith, Kath; Abu Dawud, Raed; Jones, Mark; Matin, Maryam; Gokhale, Paul; Draper, Jonathan; Andrews, Peter W

    2005-11-01

    Human embryonic stem cell (HESC) lines vary in their characteristics and behaviour not only because they are derived from genetically outbred populations, but also because they may undergo progressive adaptation upon long-term culture in vitro. Such adaptation may reflect selection of variants with altered propensity for survival and retention of an undifferentiated phenotype. Elucidating the mechanisms involved will be important for understanding normal self-renewal and commitment to differentiation and for validating the safety of HESC-based therapy. We have investigated this process of adaptation at the cellular and molecular levels through a comparison of early passage (normal) and late passage (adapted) sublines of a single HESC line, H7. To account for spontaneous differentiation that occurs in HESC cultures, we sorted cells for SSEA3, which marks undifferentiated HESC. We show that the gene expression programmes of the adapted cells partially reflected their aberrant karyotype, but also resulted from a failure in X-inactivation, emphasizing the importance in adaptation of karyotypically silent epigenetic changes. On the basis of growth potential, ability to re-initiate ES cultures and global transcription profiles, we propose a cellular differentiation hierarchy for maintenance cultures of HESC: normal SSEA3+ cells represent pluripotent stem cells. Normal SSEA3- cells have exited this compartment, but retain multilineage differentiation potential. However, adapted SSEA3+ and SSEA3- cells co-segregate within the stem cell territory, implying that adaptation reflects an alteration in the balance between self-renewal and differentiation. As this balance is also an essential feature of cancer, the mechanisms of culture adaptation may mirror those of oncogenesis and tumour progression. PMID:16159889

  11. Protein, free amino acid, phenloic, ß-carotene, and lycopene content, and antioxidative and cancer cell inhibitory effects of 12 greenhouse-grown commercial cherry tomato varieties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The content of water, free amino acids, amino acid metabolites, crude protein, the carotene pigments ß-carotene and lycopene, and 9 characterized and 2 incompletely characterized individual phenolic (flavonoid) compounds of 12 greenhouse-grown cherry tomato varieties of various colors (green, yellow...

  12. Isolation and purification of self-renewable human neural stem cells for cell therapy in experimental model of ischemic stroke.

    PubMed

    Azevedo-Pereira, Ricardo L; Daadi, Marcel M

    2013-01-01

    Human embryonic stem cells (hESCs) are pluripotent with a strong self-renewable ability making them a virtually unlimited source of neural cells for structural repair in neurological disorders. Currently, hESCs are one of the most promising cell sources amenable for commercialization of off-shelf cell therapy products. However, along with this strong proliferative capacity of hESCs comes the tumorigenic potential of these cells after transplantation. Thus, the isolation and purification of a homogeneous, population of neural stem cells (hNSCs) are of paramount importance to avoid tumor formation in the host brain. This chapter describes the isolation, neuralization, and long-term perpetuation of hNSCs derived from hESCs through use of specific mitogenic growth factors and the preparation of hNSCs for transplantation in an experimental model of stroke. Additionally, we describe methods to analyze the stroke and size of grafts using magnetic resonance imaging and Osirix software, and neuroanatomical tracing procedures to study axonal remodeling after stroke and cell transplantation.

  13. Epigenetically Reprogramming of Human Embryonic Stem Cells by 3-Deazaneplanocin A and Sodium Butyrate

    PubMed Central

    Azghadi, Soheila; Clark, Amander T.

    2011-01-01

    Objectives: Infertility affects about 6.1 million women aged 15-44 in the United States. The leading cause of infertility in women is quantitative and qualitative defects in human germ-cell development (these sentences are not mentioned in introduction so it is not correct to mention in abstract, you can omit). Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of developing blastocysts and have a broad clinical potential. hESCs have been classified into three classes based on their epigenetic state. The goal of this study was to epigenetically reprogram Class II and Class III cell lines to Class I (naïve state), and to in vitro differentiation of potent hESCs to primordial germ cells (PGCs). Methods: Recent evidence suggests that 3-deazaneplanocin A (DZNep) is a global histone methylation inhibitor which selectively inhibits trimethylation of lysine 27 on histone H3K27, and it is an epigenetic therapeutic for cancer. The characteristics of DZNep lead us to hypothesize that it is a good candidate to epigenetically reprogram hESCs to the Class I. Additionally, we used sodium butyrate (NaBu) shown in previous studies to up-regulate the expression of germ cell specific markers (these sentences should be come in introduction). Results: We used these two drugs to produce epigenetically stable hESC lines. hESC lines are an appropriate system for disease modeling and understanding developmental stages, therefore producing stable stem cell lines may have an outstanding impact in different research fields such as preventive medicine. Conclusions: X-Chromosome inactivation has been used as a tool to follow the reprogramming process. We have used immunostaining and western blot as methods to follow this reprogramming qualitatively and quantitatively. PMID:21603011

  14. Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells.

    PubMed

    Irwin, Elizabeth F; Gupta, Rohini; Dashti, Derek C; Healy, Kevin E

    2011-10-01

    We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR™1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel™-coated substrates. Mechanistically, it was resolved that bovine serum albumin (BSA) in the mTeSR™1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g., peptides, proteins, or Matrigel™) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion.

  15. 75 FR 13137 - National Institutes of Health Guidelines for Human Stem Cell Research

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-18

    ... Cell Research SUMMARY: The National Institutes of Health (NIH) is extending the public comment period on a revision to the definition of human embryonic stem cells (hESCs) in the ``National Institutes of Health Guidelines for Human Stem Cell Research'' (Guidelines). Due to a technical problem,...

  16. 75 FR 8085 - National Institutes of Health Guidelines for Human Stem Cell Research

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-23

    ... Cell Research SUMMARY: The National Institutes of Health (NIH) is requesting public comment on a revision to the definition of human embryonic stem cells (hESCs) in the ``National Institutes of Health Guidelines for Human Stem Cell Research'' (Guidelines). On July 7, 2009, NIH issued Guidelines (...

  17. Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations

    PubMed Central

    Titmarsh, Drew; Krömer, Jens O.; Kao, Li-Pin; Nielsen, Lars; Wolvetang, Ernst; Cooper-White, Justin

    2014-01-01

    As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in

  18. Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations.

    PubMed

    Turner, Jennifer; Quek, Lake-Ee; Titmarsh, Drew; Krömer, Jens O; Kao, Li-Pin; Nielsen, Lars; Wolvetang, Ernst; Cooper-White, Justin

    2014-01-01

    As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in

  19. The metabolome regulates the epigenetic landscape during naïve to primed human embryonic stem cell transition

    PubMed Central

    Sperber, Henrik; Mathieu, Julie; Wang, Yuliang; Ferreccio, Amy; Hesson, Jennifer; Xu, Zhuojin; Fischer, Karin A.; Devi, Arikketh; Detraux, Damien; Gu, Haiwei; Battle, Stephanie L.; Showalter, Megan; Valensisi, Cristina; Bielas, Jason H.; Ericson, Nolan G.; Margaretha, Lilyana; Robitaille, Aaron M.; Margineantu, Daciana; Fiehn, Oliver; Hockenbery, David; Blau, C. Anthony; Raftery, Daniel; Margolin, Adam; Hawkins, R. David; Moon, Randall T.; Ware, Carol B.; Ruohola-Baker, Hannele

    2015-01-01

    For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans display two stable yet epigenetically distinct states of pluripotency, naïve and primed. We now show that nicotinamide-N-methyl transferase (NNMT) and metabolic state regulate pluripotency in hESCs. Specifically, in naïve hESCs NNMT and its enzymatic product 1-methylnicotinamide (1-MNA) are highly upregulated, and NNMT is required for low SAM levels and H3K27me3 repressive state. NNMT consumes SAM in naïve cells, making it unavailable for histone methylation that represses Wnt and activates HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development. PMID:26571212

  20. Stem cells to pancreatic beta-cells: new sources for diabetes cell therapy.

    PubMed

    Guo, Tingxia; Hebrok, Matthias

    2009-05-01

    The number of patients worldwide suffering from the chronic disease diabetes mellitus is growing at an alarming rate. Insulin-secreting beta-cells in the islet of Langerhans are damaged to different extents in diabetic patients, either through an autoimmune reaction present in type 1 diabetic patients or through inherent changes within beta-cells that affect their function in patients suffering from type 2 diabetes. Cell replacement strategies via islet transplantation offer potential therapeutic options for diabetic patients. However, the discrepancy between the limited number of donor islets and the high number of patients who could benefit from such a treatment reflects the dire need for renewable sources of high-quality beta-cells. Human embryonic stem cells (hESCs) are capable of self-renewal and can differentiate into components of all three germ layers, including all pancreatic lineages. The ability to differentiate hESCs into beta-cells highlights a promising strategy to meet the shortage of beta-cells. Here, we review the different approaches that have been used to direct differentiation of hESCs into pancreatic and beta-cells. We will focus on recent progress in the understanding of signaling pathways and transcription factors during embryonic pancreas development and how this knowledge has helped to improve the methodology for high-efficiency beta-cell differentiation in vitro.

  1. Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

    PubMed Central

    MacArthur, Chad C.; Xue, Haipeng; Van Hoof, Dennis; Lieu, Pauline T.; Dudas, Miroslav; Fontes, Andrew; Swistowski, Andrzej; Touboul, Thomas; Seerke, Rina; Laurent, Louise C.; Loring, Jeanne F.; German, Michael S.; Zeng, Xianmin; Rao, Mahendra S.; Lakshmipathy, Uma; Chesnut, Jonathan D.

    2012-01-01

    Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5′ and 3′ of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken β actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs. PMID:21699412

  2. Scalable Stirred-Suspension Bioreactor Culture of Human Pluripotent Stem Cells

    PubMed Central

    Kehoe, Daniel E.; Jing, Donghui; Lock, Lye T.

    2010-01-01

    Advances in stem cell biology have afforded promising results for the generation of various cell types for therapies against devastating diseases. However, a prerequisite for realizing the therapeutic potential of stem cells is the development of bioprocesses for the production of stem cell progeny in quantities that satisfy clinical demands. Recent reports on the expansion and directed differentiation of human embryonic stem cells (hESCs) in scalable stirred-suspension bioreactors (SSBs) demonstrated that large-scale production of therapeutically useful hESC progeny is feasible with current state-of-the-art culture technologies. Stem cells have been cultured in SSBs as aggregates, in microcarrier suspension and after encapsulation. The various modes in which SSBs can be employed for the cultivation of hESCs and human induced pluripotent stem cells (hiPSCs) are described. To that end, this is the first account of hiPSC cultivation in a microcarrier stirred-suspension system. Given that cultured stem cells and their differentiated progeny are the actual products used in tissue engineering and cell therapies, the impact of bioreactor's operating conditions on stem cell self-renewal and commitment should be considered. The effects of variables specific to SSB operation on stem cell physiology are discussed. Finally, major challenges are presented which remain to be addressed before the mainstream use of SSBs for the large-scale culture of hESCs and hiPSCs. PMID:19739936

  3. Scalable stirred-suspension bioreactor culture of human pluripotent stem cells.

    PubMed

    Kehoe, Daniel E; Jing, Donghui; Lock, Lye T; Tzanakakis, Emmanuel S

    2010-02-01

    Advances in stem cell biology have afforded promising results for the generation of various cell types for therapies against devastating diseases. However, a prerequisite for realizing the therapeutic potential of stem cells is the development of bioprocesses for the production of stem cell progeny in quantities that satisfy clinical demands. Recent reports on the expansion and directed differentiation of human embryonic stem cells (hESCs) in scalable stirred-suspension bioreactors (SSBs) demonstrated that large-scale production of therapeutically useful hESC progeny is feasible with current state-of-the-art culture technologies. Stem cells have been cultured in SSBs as aggregates, in microcarrier suspension and after encapsulation. The various modes in which SSBs can be employed for the cultivation of hESCs and human induced pluripotent stem cells (hiPSCs) are described. To that end, this is the first account of hiPSC cultivation in a microcarrier stirred-suspension system. Given that cultured stem cells and their differentiated progeny are the actual products used in tissue engineering and cell therapies, the impact of bioreactor's operating conditions on stem cell self-renewal and commitment should be considered. The effects of variables specific to SSB operation on stem cell physiology are discussed. Finally, major challenges are presented which remain to be addressed before the mainstream use of SSBs for the large-scale culture of hESCs and hiPSCs.

  4. Inhibition of IKK/NF-κB Signaling Enhances Differentiation of Mesenchymal Stromal Cells from Human Embryonic Stem Cells

    PubMed Central

    Deng, Peng; Zhou, Chenchen; Alvarez, Ruth; Hong, Christine; Wang, Cun-Yu

    2016-01-01

    Summary Embryonic stem cell-derived mesenchymal stromal cells (MSCs; also known as mesenchymal stem cells) represent a promising source for bone regenerative medicine. Despite remarkable advances in stem cell biology, the molecular mechanism regulating differentiation of human embryonic stem cells (hESCs) into MSCs remains poorly understood. Here, we report that inhibition of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling enhances differentiation of hESCs into MSCs by expediting the loss of pluripotent markers and increasing the expression of MSC surface markers. In addition, a significantly higher quantity of MSCs was produced from hESCs with IKK/NF-κB suppression. These isolated MSCs displayed evident multipotency with capacity to terminally differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and to form bone in vivo. Collectively, our data provide important insights into the role of NF-κB in mesenchymal lineage specification during hESC differentiation, suggesting that IKK inhibitors could be utilized as an adjuvant in generating MSCs for cell-mediated therapies. PMID:26972683

  5. Challenges and strategies for generating therapeutic patient-specific hemangioblasts and hematopoietic stem cells from human pluripotent stem cells

    PubMed Central

    PETERS, ANN; BURRIDGE, PAUL W.; PRYZHKOVA, MARINA V.; LEVINE, MICHAL A.; PARK, TEA-SOON; ROXBURY, CHRISTOPHER; YUAN, XUAN; PÉAULT, BRUNO; ZAMBIDIS, ELIAS T.

    2012-01-01

    Recent characterization of hemangioblasts differentiated from human embryonic stem cells (hESC) has further confirmed evidence from murine, zebrafish and avian experimental systems that hematopoietic and endothelial lineages arise from a common progenitor. Such progenitors may provide a valuable resource for delineating the initial developmental steps of human hemato-endotheliogenesis, which is a process normally difficult to study due to the very limited accessibility of early human embryonic/fetal tissues. Moreover, efficient hemangioblast and hematopoietic stem cell (HSC) generation from patient-specific pluripotent stem cells has enormous potential for regenerative medicine, since it could lead to strategies for treating a multitude of hematologic and vascular disorders. However, significant scientific challenges remain in achieving these goals, and the generation of transplantable hemangioblasts and HSC derived from hESC currently remains elusive. Our previous work has suggested that the failure to derive engraftable HSC from hESC is due to the fact that current methodologies for differentiating hESC produce hematopoietic progenitors developmentally similar to those found in the human yolk sac, and are therefore too immature to provide adult-type hematopoietic reconstitution. Herein, we outline the nature of this challenge and propose targeted strategies for generating engraftable human pluripotent stem cell-derived HSC from primitive hemangioblasts using a developmental approach. We also focus on methods by which reprogrammed somatic cells could be used to derive autologous pluripotent stem cells, which in turn could provide unlimited sources of patient-specific hemangioblasts and HSC. PMID:20563986

  6. Stem cells in cardiac repair.

    PubMed

    Henning, Robert J

    2011-01-01

    Myocardial infarction is the leading cause of death among people in industrialized nations. Although the heart has some ability to regenerate after infarction, myocardial restoration is inadequate. Consequently, investigators are currently exploring the use of human embryonic stem cells (hESCs), skeletal myoblasts and adult bone marrow stem cells to limit infarct size. hESCs are pluripotent cells that can regenerate myocardium in infarcted hearts, attenuate heart remodeling and contribute to left ventricle (LV) systolic force development. Since hESCs can form heart teratomas, investigators are differentiating hESCs toward cardiac progenitor cells prior to transplantation into hearts. Large quantities of hESCs cardiac progenitor cells, however, must be generated, immune rejection must be prevented and grafts must survive over the long term to significantly improve myocardial performance. Transplanted autologous skeletal myoblasts can survive in infarcted myocardium in small numbers, proliferate, differentiate into skeletal myofibers and increase the LV ejection fraction. These cells, however, do not form electromechanical connections with host cardiomyocytes. Consequently, electrical re-entry can occur and cause cardiac arrhythmias. Autologous bone marrow mononuclear cells contain hematopoietic and mesenchymal stem cells. In several meta-analyses, patients with coronary disease who received autologous bone marrow cells by intracoronary injection show significant 3.7% (range: 1.9-5.4%) increases in LV ejection fraction, decreases in LV end-systolic volume of -4.8 ml (range: -1.4 to -8.2 ml) and reductions in infarct size of 5.5% (-1.9 to -9.1%), without experiencing arrhythmias. Bone marrow cells appear to release biologically active factors that limit myocardial damage. Unfortunately, bone marrow cells from patients with chronic diseases propagate poorly and can die prematurely. Substantial challenges must be addressed and resolved to advance the use of stem cells

  7. Isolation and transplantation of corneal endothelial cell-like cells derived from in-vitro-differentiated human embryonic stem cells.

    PubMed

    Zhang, Kai; Pang, Kunpeng; Wu, Xinyi

    2014-06-15

    The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell-conditioned medium. Within 1 week, CEC-like cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the N-cadherin/vimentin dual-positive population enriches for CEC-like cells. The isolated CEC-like cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and seeded onto posterior acellular porcine corneal matrix lamellae to construct the CEC-like cell sheets. Pump function parameters of the CEC-like cell sheets approximated those of human donor corneas. Importantly, when the CEC-like cell sheets were transplanted into the eyes of rabbit CE dysfunction models, the corneal transparency was restored gradually. In conclusion, CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CEC biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future. PMID:24499373

  8. JMJD5 regulates cell cycle and pluripotency in human embryonic stem cells.

    PubMed

    Zhu, Hui; Hu, Shijun; Baker, Julie

    2014-08-01

    In mammalian embryos, embryonic stem cells (ESCs) and induced pluripotent cells, a shortened G1 phase is correlated with the pluripotent state. To molecularly define this phase, we compared transcripts from the shortened G1 of human ESCs (hESCs) with those from the longer G1 of derived endoderm. We identified JMJD5, a JmjC (Jumonji C) domain containing protein that, when depleted in hESCs, causes the accumulation of cells in G1 phase, loss of pluripotency, and subsequent differentiation into multiple lineages, most prominently ectoderm and trophectoderm. Furthermore, we demonstrate that the JMJD5 phenotype is caused by the upregulation of CDKN1A (p21), as depleting both JMJD5 and CDKN1A (p21) in hESCs restores the rapid G1 phase and rescues the pluripotent state. Overall, we provide genetic and biochemical evidence that the JMJD5/CDKN1A (p21) axis is essential to maintaining the short G1 phase which is critical for pluripotency in hESCs.

  9. Enriched retinal ganglion cells derived from human embryonic stem cells.

    PubMed

    Gill, Katherine P; Hung, Sandy S C; Sharov, Alexei; Lo, Camden Y; Needham, Karina; Lidgerwood, Grace E; Jackson, Stacey; Crombie, Duncan E; Nayagam, Bryony A; Cook, Anthony L; Hewitt, Alex W; Pébay, Alice; Wong, Raymond C B

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  10. Enriched retinal ganglion cells derived from human embryonic stem cells

    PubMed Central

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  11. Cryopreserved mouse fetal liver stromal cells treated with mitomycin C are able to support the growth of human embryonic stem cells

    PubMed Central

    ZHANG, WEI; HU, JIABO; MA, QUANHUI; HU, SANQIANG; WANG, YANYAN; WEN, XIANGMEI; MA, YONGBIN; XU, HONG; QIAN, HUI; XU, WENRONG

    2014-01-01

    An immortalized mouse fetal liver stromal cell line, named KM3, has demonstrated the potential to support the growth and maintenance of human embryonic stem cells (hESCs). In this study, the characteristics of KM3 cells were examined following cryopreservation at −70°C and in liquid nitrogen for 15, 30 and 60 days following treatment with 10 μg/ml mitomycin C. In addition, whether the KM3 cells were suitable for use as feeder cells to support the growth of hESCs was evaluated. The inhibition of mitosis without cell death was observed when the KM3 cells were treated with 10 μg/ml mitomycin C for 2 h. The morphology of the KM3 cells cryopreserved in liquid nitrogen for 60 days was not markedly changed, and the cell survival rate was 84.60±1.14%. By contrast, the survival rate of the KM3 cells was 66.40±2.88% following cryopreservation at −70°C for 60 days; the cells readily detached, were maintained for a shorter time, and had a reduced expression level of basic fibroblast growth factor. hESCs cultured on KM3 cells cryopreserved in liquid nitrogen for 60 days showed the typical bird’s nest structure, with clear boundaries and a differentiation rate of 16.33±2.08%. The differentiation rate of hESCs cultured on KM3 cells cryopreserved at −70°C for 60 days was 37.67±3.51%. These results indicate that the cryopreserved KM3 cells treated with mitomycin C may be directly used in the subculture of hESCs, and the effect is relatively good with −70°C short-term or liquid nitrogen cryopreservation. PMID:25120627

  12. The effect of actin disrupting agents on contact guidance of human embryonic stem cells

    PubMed Central

    Gerecht, Sharon; Bettinger, Christopher J.; Zhang, Zhitong; Borenstein, Jeffrey; Vunjak-Novakovic, Gordana; Langer, Robert

    2007-01-01

    Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that substrate nanotopography alters morpohology and proliferation of human embryonic stem cells (hESCs). Fibronectin-coated poly(di-methyl siloxane) substrates with line-grating (600 nm ridges with 600 nm spacing and 600 +/− 150 nm feature height) induced hESC alignment and elongation, mediated the organization of cytoskeletal components including actin, vimentin, and α-tubulin, and reduced proliferation. Spatial polarization of gamma tubulin complexes was also observed in response to nanotopography. Furthermore, the addition of actin disrupting agents attenuated the alignment and proliferative effects of nanotopography. These findings further demonstrate the importance of interplay between cytoskeleton and substrate interactions as a key modulator of morphological and proliferative cellular response in hESCs on nanotopography. PMID:17576011

  13. The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells.

    PubMed

    Honda, Makoto; Minami, Itsunari; Tooi, Norie; Morone, Nobuhiro; Nishioka, Hisae; Uemura, Kengo; Kinoshita, Ayae; Heuser, John E; Nakatsuji, Norio; Aiba, Kazuhiro

    2016-01-15

    Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-β 42 (Aβ42)/Aβ40 and Aβ43/Aβ40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery.

  14. Graphic Grown Up

    ERIC Educational Resources Information Center

    Kim, Ann

    2009-01-01

    It's no secret that children and YAs are clued in to graphic novels (GNs) and that comics-loving adults are positively giddy that this format is getting the recognition it deserves. Still, there is a whole swath of library card-carrying grown-up readers out there with no idea where to start. Splashy movies such as "300" and "Spider-Man" and their…

  15. Continuous Hypoxic Culturing of Human Embryonic Stem Cells Enhances SSEA-3 and MYC Levels

    PubMed Central

    Laiho, Asta; Rahkonen, Nelly; Emani, Maheswara Reddy; Viitala, Miro; Laurila, Kirsti; Sahla, Roosa; Lund, Riikka; Lähdesmäki, Harri; Jaakkola, Panu; Lahesmaa, Riitta

    2013-01-01

    Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However, it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9, HS401 and HS360) on short (2 hours), intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore, the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs, were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover, transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further, MYC protein expression in hypoxia was affected by silencing HIF2α, but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state. PMID:24236059

  16. Translating human embryonic stem cells from 2-dimensional to 3-dimensional cultures in a defined medium on laminin- and vitronectin-coated surfaces.

    PubMed

    Heng, Boon Chin; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul; Cool, Simon M; Birch, William R; Oh, Steve Kah-Weng

    2012-07-01

    While defining the environment for human embryonic stem cell (hESC) culture on 2-dimensional (2D) surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a 3-dimensional (3D) system, thus enabling better control, automation, and volumetric scale-up in bioreactors. The current study describes a system with defined conditions that are capable of supporting the long-term 2D culture of hESCs and the transposing of these conditions to 3D microcarrier (MC) cultures. Vitronectin (VN) and laminin (LN) were chosen as matrices for the long-term propagation of hESCs in a defined culture medium (STEMPRO(®)) for conventional 2D culture. Adsorption of these proteins onto 2D tissue culture polystyrene (TCPS) indicated that surface density saturation of 510 and 850 ng/cm(2) for VN and LN, respectively, was attained above 20 μg/mL deposition solution concentration. Adsorption of these proteins onto spherical (97±10 μm), polystyrene MC followed a similar trend and coating surface densities of 450 and 650 ng/cm(2) for VN and LN, respectively, were used to support hESC propagation. The long-term expansion of hESCs was equally successful on TCPS and MC, with consistently high expression (>90%) of pluripotent markers (OCT-4, MAB-84, and TRA-1-60) over 20 passages and maintenance of karyotypic normality. The average fold increase in cell numbers on VN-coated MC per serial passage was 8.5±1.0, which was similar to LN-coated MC (8.5±0.9). Embryoid body differentiation assays and teratoma formation confirmed that hESCs retained the ability to differentiate into lineages of all 3 germ layers, thus demonstrating the first translation to a fully defined MC-based environment for the expansion of hESCs.

  17. Topoisomerase I inhibitor, camptothecin, induces apoptogenic signaling in human embryonic stem cells.

    PubMed

    García, Carolina Paola; Videla Richardson, Guillermo Agustín; Romorini, Leonardo; Miriuka, Santiago Gabriel; Sevlever, Gustavo Emilio; Scassa, María Elida

    2014-03-01

    Embryonic stem cells (ESCs) need to maintain their genomic integrity in response to DNA damage to safeguard the integrity of the organism. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and, if not repaired correctly, they can lead to cell death, genomic instability and cancer. How human ESCs (hESCs) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. In the present study we aim to determine the hESC response to the DSB inducing agent camptothecin (CPT). We find that hESCs are hypersensitive to CPT, as evidenced by high levels of apoptosis. CPT treatment leads to DNA-damage sensor kinase (ATM and DNA-PKcs) phosphorylation on serine 1981 and serine 2056, respectively. Activation of ATM and DNA-PKcs was followed by histone H2AX phosphorylation on Ser 139, a sensitive reporter of DNA damage. Nuclear accumulation and ATM-dependent phosphorylation of p53 on serine 15 were also observed. Remarkably, hESC viability was further decreased when ATM or DNA-PKcs kinase activity was impaired by the use of specific inhibitors. The hypersensitivity to CPT treatment was markedly reduced by blocking p53 translocation to mitochondria with pifithrin-μ. Importantly, programmed cell death was achieved in the absence of the cyclin dependent kinase inhibitor, p21(Waf1), a bona fide p53 target gene. Conversely, differentiated hESCs were no longer highly sensitive to CPT. This attenuated apoptotic response was accompanied by changes in cell cycle profile and by the presence of p21(Waf1). The results presented here suggest that p53 has a key involvement in preventing the propagation of damaged hESCs when genome is threatened. As a whole, our findings support the concept that the phenomenon of apoptosis is a prominent player in normal embryonic development.

  18. Retroviral transduction of hematopoietic progenitors derived from human embryonic stem cells.

    PubMed

    Menendez, Pablo; Wang, Lisheng; Cerdan, Chantal; Bhatia, Mickie

    2006-01-01

    It has been recently identified that cytokines and BMP-4 promote hematopoiesis from human embryonic stem cells (hESC) and that, before hematopoietic commitment, a rare subpopulation of cells lacking CD45, but expressing PECAM-1, Flk-1, and VE-cadherin (hereinafter termed CD45(neg)PFV precursors), are exclusively responsible for hematopoietic cell fate on cytokine stimulation. Efficient strategies to stably transduce these hematopoietic precursors specifically generated from hESCs would provide a novel and desirable tool to study hematopoietic development through the introduction and characterization of candidate genes suspected to regulate self-renewal processes of hESC-derived hematopoietic cells or dynamically track hESC-derived hematopoietic stem cells in vivo. To date, only transient transfection and stable transduction using lentiviral vectors have been reported in undifferentiated hESC followed by random and spontaneous differentiation into different cell types. However, protocols for stable transduction of hematopoietic progenitors prospectively derived from hESC need to be developed yet. In the present chapter, we described detailed methods on the recently characterized and optimized GALV-pseudotyped retroviral gene transfer strategy to stably transduce the hematopoietic progenitor cells prospectively derived from CD45(neg)PFV hemogenic precursors as a vital tool to study hematopoietic development and to characterize candidate genes suspected to eventually confer robust and sustained repopulating ability to hESC-derived hematopoietic cells.

  19. 1.7eV Al0.2Ga0.8As solar cells epitaxially grown on silicon by SSMBE using a superlattice and dislocation filters

    NASA Astrophysics Data System (ADS)

    Onno, Arthur; Wu, Jiang; Jiang, Qi; Chen, Siming; Tang, Mingchu; Maidaniuk, Yurii; Benamara, Mourad; Mazur, Yuriy I.; Salamo, Gregory J.; Harder, Nils-Peter; Oberbeck, Lars; Liu, Huiyin

    2016-03-01

    Lattice-mismatched 1.7eV Al0.2Ga0.8As photovoltaic solar cells have been monolithically grown on Si substrates using Solid Source Molecular Beam Epitaxy (SSMBE). As a consequence of the 4%-lattice-mismatch, threading dislocations (TDs) nucleate at the interface between the Si substrate and III-V epilayers and propagate to the active regions of the cell. There they act as recombination centers and degrade the performances of the cell. In our case, direct AlAs/GaAs superlattice growth coupled with InAlAs/AlAs strained layer superlattice (SLS) dislocation filter layers (DFLSs) have been used to reduce the TD density from 1×109cm-2 to 1(+/-0.2)×107cm-2. Lattice-matched Al0.2Ga0.8As cells have also been grown on GaAs as a reference. The best cell grown on silicon exhibits a Voc of 964mV, compared with a Voc of 1128mV on GaAs. Fill factors of respectively 77.6% and 80.2% have been calculated. Due to the lack of an anti-reflection coating and the non-optimized architecture of the devices, relatively low Jsc have been measured: 7.30mA.cm-2 on Si and 6.74mA.cm-2 on GaAs. The difference in short-circuit currents is believed to be caused by a difference of thickness between the samples due to discrepancies in the calibration of the MBE prior to each growth. The bandgap-voltage offset of the cells, defined as Eg/q-Voc, is relatively high on both substrates with 736mV measured on Si versus 572mV on GaAs. The non-negligible TD density partly explains this result on Si. On GaAs, non-ideal growth conditions are possibly responsible for these suboptimal performances.

  20. Functional Characterization and Expression Profiling of Human Induced Pluripotent Stem Cell- and Embryonic Stem Cell-Derived Endothelial Cells

    PubMed Central

    Li, Zongjin; Hu, Shijun; Ghosh, Zhumur; Han, Zhongchao

    2011-01-01

    With regard to human induced pluripotent stem cells (hiPSCs), in which adult cells are reprogrammed into embryonic-like cells using defined factors, their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study, hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method, and CD31+ cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the “distances” among hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and HUVECs. We further conclude that hiPSCs can differentiate into functional endothelial cells, but with limited expansion potential compared with hESC-ECs; thus, extensive studies should be performed to explore the cause and extent of such differences before clinical application of hiPSC-ECs can begin. PMID:21235328

  1. Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD) for monogenic disorder.

    PubMed

    Hmadcha, Abdelkrim; Aguilera, Yolanda; Lozano-Arana, Maria Dolores; Mellado, Nuria; Sánchez, Javier; Moya, Cristina; Sánchez-Palazón, Luis; Palacios, Jose; Antiñolo, Guillermo; Soria, Bernat

    2016-05-01

    From 106 human blastocyts donate for research after in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for monogenetic disorder, 3 human embryonic stem cells (hESCs) HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15)(q34.3;q14) detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were registered in the Spanish Stem Cell Bank. PMID:27346196

  2. Gene expression signatures affected by alcohol-induced DNA methylomic deregulation in human embryonic stem cells

    PubMed Central

    Kim, Hyun-Sung; Hoang, Michael; Tu, Thanh G.; Elie, Omid; Lee, Connie; Vu, Catherine; Horvath, Steve; Spigelman, Igor; Kim, Yong

    2014-01-01

    Stem cells, especially human embryonic stem cells (hESCs), are useful models to study molecular mechanisms of human disorders that originate during gestation. Alcohol (ethanol, EtOH) consumption during pregnancy causes a variety of prenatal and postnatal disorders collectively referred to as fetal alcohol spectrum disorders (FASDs). To better understand the molecular events leading to FASDs, we performed a genome-wide analysis of EtOH's effects on the maintenance and differentiation of hESCs in culture. Gene Co-expression Network Analysis showed significant alterations in gene profiles of EtOH-treated differentiated or undifferentiated hESCs, particularly those associated with molecular pathways for metabolic processes, oxidative stress, and neuronal properties of stem cells. A genome-wide DNA methylome analysis revealed widespread EtOH-induced alterations with significant hypermethylation of many regions of chromosomes. Undifferentiated hESCs were more vulnerable to EtOH's effect than their differentiated counterparts, with methylation on the promoter regions of chromosomes 2, 16 and 18 in undifferentiated hESCs most affected by EtOH exposure. Combined transcriptomic and DNA methylomic analysis produced a list of differentiation-related genes dysregulated by EtOH-induced DNA methylation changes, which likely play a role in EtOH-induced decreases in hESC pluripotency. DNA sequence motif analysis of genes epigenetically altered by EtOH identified major motifs representing potential binding sites for transcription factors. These findings should help in deciphering the precise mechanisms of alcohol-induced teratogenesis. PMID:24751885

  3. A novel immortalized human endometrial stromal cell line with normal progestational response.

    PubMed

    Krikun, Graciela; Mor, Gil; Alvero, Ayesha; Guller, Seth; Schatz, Frederick; Sapi, Eva; Rahman, Mizanur; Caze, Rebeca; Qumsiyeh, Mazin; Lockwood, Charles J

    2004-05-01

    Obtaining primary human endometrial stromal cells (HESCs) for in vitro studies is limited by the scarcity of adequate human material and the inability to passage these cells in culture for long periods. Immortalization of these cells would greatly facilitate studies; however, the process of immortalization often results in abnormal karyotypes and aberrant functional characteristics. To meet this need, we have introduced telomerase into cultured HESCs to prevent the normal shortening of telomeres observed in adult somatic cells during mitosis. We have now developed and analyzed a newly immortalized HESC line that contains no clonal chromosomal structural or numerical abnormalities. In addition, when compared with the primary unpassaged parent cells, the new cell line displayed similar biochemical endpoints after treatment with ovarian steroids. Classical decidualization response to estradiol plus medroxyprogesterone acetate were seen in both morphologically, and progestin was seen to induce or regulate the expression of IGF binding protein-1, fibronectin, prolactin, tissue factor, plasminogen activator inhibitor-1, and Fas/Fas ligand. In summary, an immortalized HESC line has been developed that is karyotypically, morphologically, and phenotypically similar to the primary parent cells, and it is a powerful and consistent resource for in vitro work.

  4. Thermoresponsive worms for expansion and release of human embryonic stem cells.

    PubMed

    Chen, Xiaoli; Prowse, Andrew B J; Jia, Zhongfan; Tellier, Helena; Munro, Trent P; Gray, Peter P; Monteiro, Michael J

    2014-03-10

    The development of robust suspension cultures of human embryonic stem cells (hESCs) without the use of cell membrane disrupting enzymes or inhibitors is critical for future clinical applications in regenerative medicine. We have achieved this by using long, flexible, and thermoresponsive polymer worms decorated with a recombinant vitronectin subdomain that bridge hESCs, aiding in hESC's natural ability to form embryoid bodies (EBs) and satisfying their inherent requirement for cell-cell and cell-extracellular matrix contact. When the EBs reached an optimal upper size where cytokine and nutrient penetration becomes limiting, these long and flexible polymer worms facilitated EB breakdown via a temperature shift from 37 to 25 °C. The thermoresponsive nature of the worms enabled a cyclical dissociation and propagation of the cells. Repeating the process for three cycles (over eighteen days) provided a >30-fold expansion in cell number while maintaining pluripotency, thereby providing a simple, nondestructive process for the 3D expansion of hESC. PMID:24571238

  5. A Regulatory Network Involving β-Catenin, e-Cadherin, PI3k/Akt, and Slug Balances Self-Renewal and Differentiation of Human Pluripotent Stem Cells In Response to Wnt Signaling.

    PubMed

    Huang, Tyng-Shyan; Li, Li; Moalim-Nour, Lilian; Jia, Deyong; Bai, Jian; Yao, Zemin; Bennett, Steffany A L; Figeys, Daniel; Wang, Lisheng

    2015-05-01

    The mechanisms underlying disparate roles of the canonical Wnt signaling pathway in maintaining self-renewal or inducing differentiation and lineage specification in embryonic stem cells (ESCs) are not clear. In this study, we provide the first demonstration that self-renewal versus differentiation of human ESCs (hESCs) in response to Wnt signaling is predominantly determined by a two-layer regulatory circuit involving β-catenin, E-cadherin, PI3K/Akt, and Slug in a time-dependent manner. Short-term upregulation of β-catenin does not lead to the activation of T-cell factor (TCF)-eGFP Wnt reporter in hESCs. Instead, it enhances E-cadherin expression on the cell membrane, thereby enhancing hESC self-renewal through E-cadherin-associated PI3K/Akt signaling. Conversely, long-term Wnt activation or loss of E-cadherin intracellular β-catenin binding domain induces TCF-eGFP activity and promotes hESC differentiation through β-catenin-induced upregulation of Slug. Enhanced expression of Slug leads to a further reduction of E-cadherin that serves as a β-catenin "sink" sequestering free cytoplasmic β-catenin. The formation of such a framework reinforces hESCs to switch from a state of temporal self-renewal associated with short-term Wnt/β-catenin activation to definitive differentiation. Stem Cells 2015;33:1419-1433.

  6. Trends in the geographic distribution of human embryonic stem-cell research.

    PubMed

    Levine, Aaron

    2004-09-01

    Human embryonic stem-cell (hESC) research offers substantial potential benefits but has generated politically influential controversies and, in the United States, funding restrictions. Some observers fear the United States has been falling behind nations more permissive in this field, but policy debate has remained largely anecdotal. This study reports citation data indicating that the share of hESC research publications credited to the United States in the six years following the introduction of key technologies was significantly less than in five less contentious biomedical-research areas. The United States share of hESC publications fell sharply in 2003 and remained near this reduced level in 2004. Putative explanations are reviewed and several implications discussed.

  7. Human embryonic stem cell research and Proposition 71. Reflections on California’s response to federal policy.

    PubMed

    Burgin, Eileen

    2010-09-01

    In response to former President George W. Bush's funding limitations on human embryonic stem cell (hESC) research, California voters in 2004 passed Proposition 71, the most expansive state-funded medical research initiative in United States history. This study examines California's experiment in the life sciences, a particularly fitting analysis now as President Barack Obama has freed up additional federal funding for hESC research. In addition to exploring the general pitfalls of states, rather than the federal government, serving as principal players on hESC science and the perceived flaws in California's program, the analysis considers the strengths of state activism and of California's enterprise. On balance, given the Bush administration's policy on hESC research, the U.S. benefitted from state innovation. Moreover, even with the new federal regulatory policy on hESC research, California should be able to mesh its program with the federal initiative and remain a prime mover in this arena. The essay draws on informal interviews with key actors in California and on Capitol Hill in 2008 and 2009.

  8. Human embryonic stem cell research and Proposition 71. Reflections on California’s response to federal policy.

    PubMed

    Burgin, Eileen

    2010-09-01

    In response to former President George W. Bush's funding limitations on human embryonic stem cell (hESC) research, California voters in 2004 passed Proposition 71, the most expansive state-funded medical research initiative in United States history. This study examines California's experiment in the life sciences, a particularly fitting analysis now as President Barack Obama has freed up additional federal funding for hESC research. In addition to exploring the general pitfalls of states, rather than the federal government, serving as principal players on hESC science and the perceived flaws in California's program, the analysis considers the strengths of state activism and of California's enterprise. On balance, given the Bush administration's policy on hESC research, the U.S. benefitted from state innovation. Moreover, even with the new federal regulatory policy on hESC research, California should be able to mesh its program with the federal initiative and remain a prime mover in this arena. The essay draws on informal interviews with key actors in California and on Capitol Hill in 2008 and 2009. PMID:21761982

  9. Monitoring the differentiation and migration patterns of neural cells derived from human embryonic stem cells using a microfluidic culture system.

    PubMed

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-06-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

  10. MicroRNAs regulate p21(Waf1/Cip1) protein expression and the DNA damage response in human embryonic stem cells.

    PubMed

    Dolezalova, Dasa; Mraz, Marek; Barta, Tomas; Plevova, Karla; Vinarsky, Vladimir; Holubcova, Zuzana; Jaros, Josef; Dvorak, Petr; Pospisilova, Sarka; Hampl, Ales

    2012-07-01

    Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA (miRNA) pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of miRNAs, including hESC-specific miRNAs such as those of the miR-302 family, miR-371-372 family, or C19MC miRNA cluster. Most importantly, we show that the hESC-enriched miRNA family miR-302 (miR-302a, miR-302b, miR-302c, and miR-302d) directly contributes to regulation of p21 expression in hESCs and, thus, demonstrate a novel function for miR-302s in hESCS. The described mechanism elucidates the role of miRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage.

  11. Sialyl-lactotetra, a novel cell surface marker of undifferentiated human pluripotent stem cells.

    PubMed

    Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E; Teneberg, Susann

    2014-07-01

    Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells.

  12. Sialyl-lactotetra, a Novel Cell Surface Marker of Undifferentiated Human Pluripotent Stem Cells*

    PubMed Central

    Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R.; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E.; Teneberg, Susann

    2014-01-01

    Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 109 cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells. PMID:24841197

  13. Potential for cell therapy in Parkinson's disease using genetically programmed human embryonic stem cell-derived neural progenitor cells.

    PubMed

    Ambasudhan, Rajesh; Dolatabadi, Nima; Nutter, Anthony; Masliah, Eliezer; Mckercher, Scott R; Lipton, Stuart A

    2014-08-15

    Neural transplantation is a promising strategy for restoring dopaminergic dysfunction and modifying disease progression in Parkinson's disease (PD). Human embryonic stem cells (hESCs) are a potential resource in this regard because of their ability to provide a virtually limitless supply of homogenous dopaminergic progenitors and neurons of appropriate lineage. The recent advances in developing robust cell culture protocols for directed differentiation of hESCs to near pure populations of ventral mesencephalic (A9-type) dopaminergic neurons has heightened the prospects for PD cell therapy. Here, we focus our review on current state-of-the-art techniques for harnessing hESC-based strategies toward development of a stem cell therapeutic for PD. Importantly, we also briefly describe a novel genetic-programming approach that may address many of the key challenges that remain in the field and that may hasten clinical translation.

  14. Cryopreservation of human embryonic stem cells by a programmed freezer with an oscillating magnetic field.

    PubMed

    Lin, Pei-Yi; Yang, Yao-Chen; Hung, Shih-Han; Lee, Sheng-Yang; Lee, Maw-Sheng; Chu, I-Ming; Hwang, Shiaw-Min

    2013-06-01

    Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, are an important source of cells for regenerative medicine. The immediate obstacles that need to be addressed are the poor cell survival rate of hESCs and their cell quality after cryopreservation. In this study, we used the Cell Alive System (CAS) which combines a programmed freezer with an oscillating magnetic field to reduce cryo-injury during the freezing process. The hESC clumps suspended in freezing medium were divided into three groups: (i) cells frozen by a conventional freezing container, Mr. Frosty and kept in a -80 °C freezer (MF); (ii) cells frozen to -32 °C by CAS, and then transferred to a -80 °C freezer (CAS); (iii) cells frozen to -32 °C by CAS, and then transferred to a pre-cooled Mr. Frosty and kept in a -80 °C freezer (CAS-MF) for overnight. All cryovials were placed in liquid nitrogen for one week, and hESCs were then thawed and cultured on feeder for 7 days. The results of alkaline phosphatase (AP) staining showed that the attachment efficiency of the cells cryopreserved by CAS and CAS-MF was significantly higher (29.0% and 44.0%) than in the MF method (7.0%). Furthermore, we confirmed the cells cryopreserved using CAS-MF could be subcultured while expressing pluripotent markers, differentiate into three germ layers, and maintain a normal karyotype. These results demonstrate that the use of CAS-MF offers an efficient method of hESC banking. PMID:23466687

  15. Induction of Human Embryonic and Induced Pluripotent Stem Cells Into Urothelium

    PubMed Central

    Osborn, Stephanie L.; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H.; Nolta, Jan

    2014-01-01

    In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium (“Uromedium”), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation. PMID:24657961

  16. Genetic Manipulation of Human Embryonic Stem Cells.

    PubMed

    Eiges, Rachel

    2016-01-01

    One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes, and studying their function, and are now being extensively implemented in human ES cells (HESCs). This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of HESCs and their applications. Detailed protocols for generating clones of genetically modified HESCs by transfection, electroporation, and infection will be described, with special emphasis on the important technical details that are required for this purpose. All protocols are equally effective in human-induced pluripotent stem (iPS) cells.

  17. Membrane permeability of the human pluripotent stem cells to Me₂SO, glycerol and 1,2-propanediol.

    PubMed

    Xu, Yanqing; Zhang, Liang; Xu, Jiandong; Wei, Yuping; Xu, Xia

    2014-05-15

    Due to the unlimited capacity of self-renew and ability to differentiate into derivatives of three germ layers, human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have a great potential in regenerative medicine. A major challenge we are facing during the long-term storage of human pluripotent stem cells with the conventional slow cooling rate is the low cell recovery rate after cryopreservation which cannot meet the requirements for the future clinical applications. Evaluating the cell membrane permeability and the corresponding activation energy of hESCs and hiPSCs for water and different cryoprotective agents (CPA), including dimethyl sulfoxide (Me2SO), 1,2-propandiol and glycerol, is important for facilitating the development of cryopreservation protocol to enhance cell recovery after the cryopreservation. The osmotically inactive volume of hESCs and hiPSCs determined using the Boyle-van't Hoff model was 0.32V0 and 0.42V0, respectively. The membrane permeability was assessed from the volume changes of cells exposed to Me2SO, 1,2-propanediol and glycerol at the temperatures ranging from 8 to 30°C. These results showed the biophysical differences between hESCs and hiPSCs. Their activation energy for water and CPAs extrapolated from the Arrhenius relationship indicated that the water transport was probably not through the channel-mediated mechanism. PMID:24780243

  18. Differences in stationary-phase cells of a commercial Saccharomyces cerevisiae wine yeast grown in aerobic and microaerophilic batch cultures assessed by electric particle analysis, light diffraction and flow cytometry.

    PubMed

    Portell, X; Ginovart, M; Carbó, R; Vives-Rego, J

    2011-01-01

    We applied electric particle analysis, light diffraction and flow cytometry to obtain information on the morphological changes during the stationary phase of Saccharomyces cerevisiae. The reported analyses of S. cerevisiae populations were obtained under two different conditions, aerobic and microaerophilic, at 27°C. The samples analysed were taken at between 20 and 50 h from the beginning of culture. To assist in the interpretation of the observed distributions a complexity index was used. The aerobically grown culture reached significantly greater cell density. Under these conditions, the cell density experienced a much lower reduction (3%) compared with the microaerophilic conditions (30%). Under aerobic conditions, the mean cell size determined by both electric particle analysis and light diffraction was lower and remained similar throughout the experiment. Under microaerophilic conditions, the mean cell size determined by electric particle analysis decreased slightly as the culture progressed through the stationary phase. Forward and side scatter distributions revealed two cell subpopulations under both growth conditions. However, in the aerobic growing culture the two subpopulations were more separated and hence easier to distinguish. The distributions obtained with the three experimental techniques were analysed using the complexity index. This analysis suggested that a complexity index is a good descriptor of the changes that take place in a yeast population in the stationary phase, and that it aids in the discussion and understanding of the implications of these distributions obtained by these experimental techniques.

  19. Effects of electron and proton irradiations on n/p and p/n GaAs cells grown by MOCVD

    NASA Technical Reports Server (NTRS)

    Weinberg, Irving; Swartz, Clifford K.; Hart, Russell E., Jr.

    1987-01-01

    State-of-the-art n/p and p/n heteroface GaAs cells, processed by metal organic chemical vapor deposition, were irradiated by 1 MeV electrons and 37 MeV protons and their performance determined as a function of fluence. It was found that the p/n cells were more radiation resistant than the n/p cells. The increased loss in the n/p cells was attributed to increases in series resistance and losses in the p-region resulting from the irradiation. The greater loss in fill factor observed for the n/p cells introduces the possibility that the presently observed superiority of the p/n cells may not be an intrinsic property of this configuration in GaAs.

  20. Science spin: iPS cell research in the news.

    PubMed

    Caulfield, T; Rachul, C

    2011-05-01

    Big scientific developments have always been spun to meet particular social agendas. We have seen it in the context of global warming, nuclear power, and genetically modified organisms. But few stories illustrate the phenomenon of spin as well as the reaction, and concomitant media coverage, that surrounded the November 2007 announcement regarding the reprogramming of skin cells to produce cells with qualities comparable to those of human embryonic stem cells (hESCs) known as induced pluripotent stem (iPS) cells.

  1. Novel Method To Differentiate Human Embryonic Stem Cells Into Dopaminergic Nerve Cells | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Institute on Drug Abuse's Development and Plasticity Section is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize novel methods to differentiate human embryonic stem cells into dopaminergic nerve cells. The invention described here is a novel method of differentiating human embryonic stem cells (hESCs) into dopaminergic nerve cells, which is preferable to the currently available dopaminergic differentiation techniques.

  2. Pax6 is a human neuroectoderm cell fate determinant

    PubMed Central

    Zhang, Xiaoqing; Huang, Cindy T.; Chen, Jing; Pankratz, Matthew T.; Xi, Jiajie; Li, Jin; Yang, Ying; LaVaute, Timothy M.; Li, Xue-Jun; Ayala, Melvin; Bondarenko, Gennadiy I.; Du, Zhong-Wei; Jin, Ying; Golos, Thaddeus G.; Zhang, Su-Chun

    2010-01-01

    SUMMARY The transcriptional regulation of neuroectoderm (NE) specification is unknown. Here we show that Pax6 is uniformly expressed in early NE cells of human fetuses and those differentiated from human embryonic stem cells (hESCs). This contrasts the later expression of Pax6 in restricted mouse brain regions. Knockdown of Pax6 blocks NE specification from hESCs. Overexpression of either Pax6a or Pax6b, but not Pax6 PD, triggers hESC differentiation. However, only Pax6a converts hESCs to NE. In contrast, neither loss nor gain of function of Pax6 affects mouse NE specification. Both Pax6a and Pax6b bind to pluripotent gene promoters but only Pax6a binds to NE genes during human NE specification. These findings indicate that Pax6 is a transcriptional determinant of the human NE and suggest that Pax6a and Pax6b coordinate with each other in determining the transition from pluripotency to the NE fate in human by differentially targeting pluripotent and NE genes. PMID:20621053

  3. Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.

    PubMed

    Schulz, Thomas C; Noggle, Scott A; Palmarini, Gail M; Weiler, Deb A; Lyons, Ian G; Pensa, Kate A; Meedeniya, Adrian C B; Davidson, Bruce P; Lambert, Nevin A; Condie, Brian G

    2004-01-01

    The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies. PMID:15579641

  4. Thymopentin enhances the generation of T-cell lineage derived from human embryonic stem cells in vitro.

    PubMed

    Zhu, Ming-Xia; Wan, Wen-Li; Li, Hai-Shen; Wang, Jing; Chen, Gui-An; Ke, Xiao-Yan

    2015-02-15

    Thymopentin is a group of biologically active peptide secreted mainly by the epithelial cells of thymic cortex and medulla. Whether it promotes T cells production from human embryonic stem cells(hESCs) in vitro remains an elusive issue. In the present study, we develop a novel strategy that enhances T-cell lineage differentiation of hESCs in collagen matrix culture by sequential cytokine cocktails treatment combined with thymopentin stimulation. We observed that approximately 30.75% cells expressed CD34 on day 14 of the cultures and expressed the surface markers of erythroid, lymphoid and myeloid lineages. The results of colony assays and gene expressions by RT-PCR analysis also demonstrated that hematopoietic progenitor cells (HPCs) derived from hESCs were capable of multi-lineage differentiation. Further study revealed that culturing with thymopentin treatment, the CD34(+)CD45RA(+)CD7(+) cells sorted from HPCs expressed T-cell-related genes, IKAROS, DNTT, TCRγ and TCRβ, and T-cell surface markers, CD3, cytoplasmic CD3, CD5, CD27, TCRγδ, CD4 and CD8. The differentiated cells produced the cytokines including IFN-γ, IL-2 and TNF-α in response to stimulation, providing the evidence for T-cell function of these cells. In conclusion, thymopentin enhances T-cell lineage differentiation from hESCs in vitro by mimicking thymus peptide environment in vivo.

  5. Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells.

    PubMed

    Yeo, Seungeun; Jeong, Sangkyun; Kim, Janghwan; Han, Jee-Soo; Han, Yong-Mahn; Kang, Yong-Kook

    2007-08-01

    Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation.

  6. Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells.

    PubMed

    Yeo, Seungeun; Jeong, Sangkyun; Kim, Janghwan; Han, Jee-Soo; Han, Yong-Mahn; Kang, Yong-Kook

    2007-08-01

    Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation. PMID:17548060

  7. Proteins secreted by embryonic stem cells activate cardiomyocytes through ligand binding pathways.

    PubMed

    LaFramboise, W A; Petrosko, P; Krill-Burger, J M; Morris, D R; McCoy, A R; Scalise, D; Malehorn, D E; Guthrie, R D; Becich, M J; Dhir, R

    2010-03-10

    Human embryonic stem cells (hESC) underlie embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2+/-5.0 ng/ml) to nerve growth factor (7.4+/-1.2pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair.

  8. Proteolytic enzymes in embryonated chicken eggs sustain the replication of egg-grown low-pathogenicity avian influenza viruses in cells in the absence of exogenous proteases

    PubMed Central

    Kandeil, Ahmed; Bagato, Ola; Zaraket, Hassan; Debeauchamp, Jennifer; Krauss, Scott; El-Shesheny, Rabeh; Webby, Richard J.; Ali, Mohamed A.; Kayali, Ghazi

    2014-01-01

    Low pathogenic influenza viruses grow readily in embryonated chicken eggs but require the addition of exogenous proteases to grow in MDCK cell culture. In this study, we found that influenza viruses propagated previously in eggs, can grow for up to two passages in cell culture without the addition of exogenous proteolytic enzymes. These results indicate that the reason for virus propagation in cells during the first two passages may be due to proteases from egg allantoic fluid carried over from egg culture. The ability of influenza viruses to grow in cells in the absence of trypsin is currently considered as a hallmark of highly pathogenic influenza viruses. Our data indicate that differentiating between high and low pathogenicity using cell culture only is not appropriate and other indicators such as sequence analysis and in-vitro pathogenicity index should be performed. PMID:24626064

  9. High-density InAs/GaAs1-xSbx quantum-dot structures grown by molecular beam epitaxy for use in intermediate band solar cells

    NASA Astrophysics Data System (ADS)

    Debnath, M. C.; Mishima, T. D.; Santos, M. B.; Cheng, Y.; Whiteside, V. R.; Sellers, I. R.; Hossain, K.; Laghumavarapu, R. B.; Liang, B. L.; Huffaker, D. L.

    2016-03-01

    InAs quantum-dot structures were grown using a GaAs1-xSbx matrix on a GaAs(001) substrate. The use of GaAs1-xSbx for the buffer and cap layers effectively suppressed coalescence between dots and significantly increased the dot density. The highest density (˜3.5 × 1011/cm2) was obtained for a nominal 3.0 monolayer deposition of InAs with an Sb composition of x = 13-14% in the GaAs1-xSbx matrix. When the Sb composition was increased to 18%, the resulting large photoluminescent red shift (˜90 meV) indicated the release of compressive strain inside the quantum dots. For x > 13%, we observed a significant decrease in photoluminescence intensity and an increase in the carrier lifetime (≥4.0 ns). This is attributed to the type-II band alignment between the quantum dots and matrix material.

  10. Encapsulation of bone morphogenic protein-2 with Cbfa1-overexpressing osteogenic cells derived from human embryonic stem cells in hydrogel accelerates bone tissue regeneration.

    PubMed

    Kim, Min Jung; Park, Ji Sun; Kim, Sinae; Moon, Sung-Hwan; Yang, Han Na; Park, Keun-Hong; Chung, Hyung-Min

    2011-08-01

    Bone tissue defects caused by trauma and disease are significant problems in orthopedic surgery. Human embryonic stem cells (hESCs) hold great promise for the treatment of bone tissue disease in regenerative medicine. In this study, we have established an effective method for the differentiation of osteogenic cells derived from hESCs using a lentiviral vector containing the transcription factor Cbfa1. Differentiation was initiated in embryoid body formation of Cbfa1-expressing hESCs, resulting in a highly purified population of osteogenic cells based on flow cytometric analysis. These cells also showed characteristics of osteogenic cells in vitro, as determined by reverse-transcription (RT)-polymerase chain reaction and immunocytochemistry using osteoblast-specific markers. We also evaluated the regenerative potential of Cbfa1-expressing cells derived from hESCs (hESC-CECs) compared with hESCs and the osteogenic effects of bone morphogenic protein-2 (BMP2) encapsulated in thermoreversible hydrogel in vivo. hESC-CECs were embedded in hydrogel constructs enriched with BMP2 to promote bone regeneration. We observed prominent mineralization and the formation of nodule-like structures using von Kossa and alizarin red S staining. In addition, the expression patterns of osteoblast-specific genes were verified by RT-polymerase chain reaction, and immunohistochemical analysis revealed that collagen type 1 and Cbfa1 were highly expressed in hESC-CECs compared with other cell types. Taken together, our results suggest that encapsulation of hESC-CECs with BMP2 in hydrogel constructs appears to be a promising method to enhance the in vitro osteoblastic differentiation and in vivo osteogenic activity of hESC-CECs.

  11. Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

    PubMed Central

    Suárez-Álvarez, Beatriz; Rodriguez, Ramón M.; Calvanese, Vincenzo; Blanco-Gelaz, Miguel A.; Suhr, Steve T.; Ortega, Francisco; Otero, Jesus; Cibelli, Jose B.; Moore, Harry; Fraga, Mario F.; López-Larrea, Carlos

    2010-01-01

    Background Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored. Methodology/Principal Findings We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation. Conclusions/Significance Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance

  12. N/P InP homojunction solar cells with an In0.53Ga0.47As contacting layer grown by liquid phase epitaxy

    NASA Technical Reports Server (NTRS)

    Shen, C. C.; Choi, K. Y.

    1989-01-01

    N/P InP homojunction solar cells with an In sub 0.53 Ga sub 0.47 As contacting layer were fabricated by liquid phase epitaxy (LPE). Electron-Beam-Induced-Current (EBIC) measurements were performed on several selected samples. It was found that the background doping level in the base region sometimes results in a deep junction, which greatly affects the cell performance.

  13. Dioxygenases of Chlorobiphenyl-Degrading Species Rhodococcus wratislaviensis G10 and Chlorophenol-Degrading Species Rhodococcus opacus 1CP Induced in Benzoate-Grown Cells and Genes Potentially Involved in These Processes.

    PubMed

    Solyanikova, I P; Borzova, O V; Emelyanova, E V; Shumkova, E S; Prisyazhnaya, N V; Plotnikova, E G; Golovleva, L A

    2016-09-01

    Dioxygenases induced during benzoate degradation by the actinobacterium Rhodococcus wratislaviensis G10 strain degrading haloaromatic compounds were studied. Rhodococcus wratislaviensis G10 completely degraded 2 g/liter benzoate during 30 h and 10 g/liter during 200 h. Washed cells grown on benzoate retained respiration activity for more than 90 days, and a high activity of benzoate dioxygenase was recorded for 10 days. Compared to the enzyme activities with benzoate, the activity of benzoate dioxygenases was 10-30% with 13 of 35 substituted benzoate analogs. Two dioxygenases capable of cleaving the aromatic ring were isolated and characterized: protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase. Catechol inhibited the activity of protocatechuate 3,4-dioxygenase. Protocatechuate did not affect the activity of catechol 1,2-dioxygenase. A high degree of identity was shown by MALDI-TOF mass spectrometry for protein peaks of the R. wratislaviensis G10 and Rhodococcus opacus 1CP cells grown on benzoate or LB. DNA from the R. wratislaviensis G10 strain was specifically amplified using specific primers to variable regions of genes coding α- and β-subunits of protocatechuate 3,4-dioxygenase and to two genes of the R. opacus 1CP coding catechol 1,2-dioxygenase. The products were 99% identical with the corresponding regions of the R. opacus 1CP genes. This high identity (99%) between the genes coding degradation of aromatic compounds in the R. wratislaviensis G10 and R. opacus 1CP strains isolated from sites of remote location (1400 km) and at different time (20-year difference) indicates a common origin of biodegradation genes of these strains and a wide distribution of these genes among rhodococci. PMID:27682171

  14. Human stem cell neuronal differentiation on silk-carbon nanotube composite

    NASA Astrophysics Data System (ADS)

    Chen, Chi-Shuo; Soni, Sushant; Le, Catherine; Biasca, Matthew; Farr, Erik; Chen, Eric Y.-T.; Chin, Wei-Chun

    2012-02-01

    Human embryonic stem cells [hESCs] are able to differentiate into specific lineages corresponding to regulated spatial and temporal signals. This unique attribute holds great promise for regenerative medicine and cell-based therapy for many human diseases such as spinal cord injury [SCI] and multiple sclerosis [MS]. Carbon nanotubes [CNTs] have been successfully used to promote neuronal differentiation, and silk has been widely applied in tissue engineering. This study aims to build silk-CNT composite scaffolds for improved neuron differentiation efficiency from hESCs. Two neuronal markers (β-III tubulin and nestin) were utilized to determine the hESC neuronal lineage differentiation. In addition, axonal lengths were measured to evaluate the progress of neuronal development. The results demonstrated that cells on silk-CNT scaffolds have a higher β-III tubulin and nestin expression, suggesting augmented neuronal differentiation. In addition, longer axons with higher density were found to associate with silk-CNT scaffolds. Our silk-CNT-based composite scaffolds can promote neuronal differentiation of hESCs. The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for SCI and MS patients.

  15. PRMT5 is required for human embryonic stem cell proliferation but not pluripotency.

    PubMed

    Gkountela, Sofia; Li, Ziwei; Chin, Chee Jia; Lee, Serena A; Clark, Amander T

    2014-04-01

    Human pluripotent stem cells (PSCs) are critical in vitro tools for understanding mechanisms that regulate lineage differentiation in the human embryo as well as a potentially unlimited supply of stem cells for regenerative medicine. Pluripotent human and mouse embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts share a similar transcription factor network to maintain pluripotency and self-renewal, yet there are considerable molecular differences reflecting the diverse environments in which mouse and human ESCs are derived. In the current study we evaluated the role of Protein arginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewal and pluripotency given its critical role in safeguarding mouse ESC pluripotency. Unlike the mouse, we discovered that PRMT5 has no role in hESC pluripotency. Using microarray analysis we discovered that a significant depletion in PRMT5 RNA and protein from hESCs changed the expression of only 78 genes, with the majority being repressed. Functionally, we discovered that depletion of PRMT5 had no effect on expression of OCT4, NANOG or SOX2, and did not prevent teratoma formation. Instead, we show that PRMT5 functions in hESCs to regulate proliferation in the self-renewing state by regulating the fraction of cells in Gap 1 (G1) of the cell cycle and increasing expression of the G1 cell cycle inhibitor P57. Taken together our data unveils a distinct role for PRMT5 in hESCs and identifies P57 as new target. PMID:24477620

  16. Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells.

    PubMed

    Butler, John T; Hall, Lisa L; Smith, Kelly P; Lawrence, Jeanne B

    2009-07-01

    The complex nuclear structure of somatic cells is important to epigenomic regulation, yet little is known about nuclear organization of human embryonic stem cells (hESC). Here we surveyed several nuclear structures in pluripotent and transitioning hESC. Observations of centromeres, telomeres, SC35 speckles, Cajal Bodies, lamin A/C and emerin, nuclear shape and size demonstrate a very different "nuclear landscape" in hESC. This landscape is remodeled during a brief transitional window, concomitant with or just prior to differentiation onset. Notably, hESC initially contain abundant signal for spliceosome assembly factor, SC35, but lack discrete SC35 domains; these form as cells begin to specialize, likely reflecting cell-type specific genomic organization. Concomitantly, nuclear size increases and shape changes as lamin A/C and emerin incorporate into the lamina. During this brief window, hESC exhibit dramatically different PML-defined structures, which in somatic cells are linked to gene regulation and cancer. Unlike the numerous, spherical somatic PML bodies, hES cells often display approximately 1-3 large PML structures of two morphological types: long linear "rods" or elaborate "rosettes", which lack substantial SUMO-1, Daxx, and Sp100. These occur primarily between Day 0-2 of differentiation and become rare thereafter. PML rods may be "taut" between other structures, such as centromeres, but clearly show some relationship with the lamina, where PML often abuts or fills a "gap" in early lamin A/C staining. Findings demonstrate that pluripotent hES cells have a markedly different overall nuclear architecture, remodeling of which is linked to early epigenomic programming and involves formation of unique PML-defined structures.

  17. Prostate tumor grown in NASA Bioreactor

    NASA Technical Reports Server (NTRS)

    2001-01-01

    This prostate cancer construct was grown during NASA-sponsored bioreactor studies on Earth. Cells are attached to a biodegradable plastic lattice that gives them a head start in growth. Prostate tumor cells are to be grown in a NASA-sponsored Bioreactor experiment aboard the STS-107 Research-1 mission in 2002. Dr. Leland Chung of the University of Virginia is the principal investigator. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: NASA and the University of Virginia.

  18. Gamete Donor Consent and Human Embryonic Stem Cell Research.

    PubMed

    Siegel, Andrew W

    2015-06-01

    There is a lack of consensus on whether the derivation and use of human embryonic stem cells (hESCs) from embryos remaining after infertility treatment morally require the informed consent of third-party gamete donors who contributed to the creation of the embryos. The principal guidelines for oversight and funding of hESC research in the United States make minimal or no demands for consent from gamete donors. In this article, I consider the arguments supporting and opposing gamete donor consent for hESC research and embryo research more broadly. I argue that it is not morally permissible to use leftover embryos in research without the informed consent of gamete donors, and that we should place restrictions on the use of existing hESC lines that may have been derived without informed consent. While the standard argument for this position relies on an appeal to gamete donors' interest in controlling what happens with their genetic material, I identify shortcomings with the standard approach and seek instead to locate the deeper moral foundations for gamete donor consent in rights to bodily integrity.

  19. High-efficiency screen-printed solar cell on edge-defined film-fed grown ribbon silicon through optimized rapid belt co-firing of contacts and high-sheet-resistance emitter

    NASA Astrophysics Data System (ADS)

    Rohatgi, Ajeet; Hilali, Mohamed M.; Nakayashiki, Kenta

    2004-04-01

    High-quality screen-printed contacts were achieved on a high-sheet-resistance emitter (˜100 Ω/sq.) using PV168 Ag paste and rapid co-firing in the belt furnace. The optimized co-firing cycle developed for a 100 Ω/sq. emitter produced 16.1% efficient 4 cm2 planar edge-defined film-fed grown (EFG) ribbon Si cells with a low series-resistance (0.8 Ω cm2), high fill factor of ˜0.77, along with very significant bulk lifetime enhancement from 3 to 100 μs. This represents the highest-efficiency screen-printed EFG Si cells with single-layer antireflection (AR) coating. These cells were fabricated using a simple process involving POCl3 diffusion for a high-sheet-resistance emitter, SiNx AR coating and rapid cofiring of Ag grid and Al-doped back-surface field in a conventional belt furnace. The rapid cofiring process also prevented junction shunting while maintaining very effective SiNx-induced hydrogen passivation of defects, resulting in an average bulk lifetime exceeding 100 μs.

  20. Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions.

    PubMed

    Álvarez, Romina S; Sacerdoti, Flavia; Jancic, Carolina; Paton, Adrienne W; Paton, James C; Ibarra, Cristina; Amaral, María M

    2016-01-01

    Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20-30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the

  1. Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions

    PubMed Central

    Álvarez, Romina S.; Sacerdoti, Flavia; Jancic, Carolina; Paton, Adrienne W.; Paton, James C.; Ibarra, Cristina; Amaral, María M.

    2016-01-01

    Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20–30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the

  2. Effects of cyclophosphamide and acrolein in organoid cultures of mouse limb bud cells grown in the presence of adult rat hepatocytes.

    PubMed

    Ghaida, J; Merker, H J

    1992-01-01

    The effects were evaluated of cyclophosphamide (CPA) and its metabolite, acrolein, on chondrogenesis in organoid cultures of mouse limb bud mesenchymal cells co-cultured with non-enzymatically isolated adult rat hepatocytes. The studies were conducted with or without the simultaneous addition of 2-mercaptoethanesulphonic acid sodium (mesna) or glutathione (GSH). Alcian blue binding assay and light and electron microscopic techniques were used. Increasing concentrations of the two compounds (bioactivated CPA, 18-180 mum; acrolein, 50-500 mum) led to a dose-dependent inhibition of chondrogenesis associated with cellular dedifferentiation and/or cytotoxicity. Addition of mesna (1 mm) or GSH (1 mm) partially protected the cultures against CPA and acrolein. However, the protective effect depended on the dose of CPA or acrolein used. A higher protection was observed with mesna than with GSH, and the effect was more pronounced with acrolein than with CPA. The morphological findings suggested that CPA and acrolein acted by different mechanisms. Bioactivated CPA primarily inhibited the differentiation process, whereas acrolein exhibited a high cytotoxic activity affecting particularly monolayer cells that normally grow on the periphery of the cultures. These findings suggest that acrolein possesses a specific mode of action directed towards this type of cell. This could be explained by the specific shape and/or behaviour of the cells (i.e. cytoskeletal arrangement, proliferation rate, migration activity, intercellular communication pattern, etc.). The results demonstrated that the cell system used was suitable for the performance of cytotoxicity and teratogenicity studies such as those conducted with CPA and acrolein.

  3. The Aberrant DNA Methylation Profile of Human Induced Pluripotent Stem Cells Is Connected to the Reprogramming Process and Is Normalized During In Vitro Culture

    PubMed Central

    Tesarova, Lenka; Simara, Pavel; Stejskal, Stanislav; Koutna, Irena

    2016-01-01

    The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications. PMID:27336948

  4. Generation of eye field/optic vesicle-like structures from human embryonic stem cells under two-dimensional and chemically defined conditions.

    PubMed

    Parvini, Maryam; Parivar, Kazem; Safari, Fatemeh; Tondar, Mahdi

    2015-03-01

    Despite the enormous progress in studying retinal cell differentiation from human embryonic stem cells (hESCs), none of the reported protocols have produced a cost-effective eye field cells with the capability to further differentiate into retinal derivatives. In this study, by drawing chemicals on our four-step differentiation strategy, we demonstrated the ability of hESCs in assembling such qualifications to follow human retinogenesis in a serum- and feeder-free adherent condition. Two-dimensional (2D) populations of eye field cells arose within early forebrain progeny upon hESCs differentiation. Gene expression analysis showed that the treatment of hESCs with a combination of selected small molecules (SMs) gave rise to the higher expressions of eye field-specific genes, PAX6, RX, and SIX3. Thereafter, a subset of cells gained the transient features of advancing retinal differentiation, including optic vesicle (OV)-like structures, which expressed MITF and CHX10 in a manner imitated in vivo human retinal development. The competency of derived cells in differentiation to retinal derivatives was further investigated. The gene analysis of the cells showed more propensity for generating retinal pigment epithelial (RPE) than neural retina (NR). The generation of OV-like structures in 2D cultures can shed light on molecular events governing retinal specification. It can also facilitate the study of human retinal development.

  5. The Aberrant DNA Methylation Profile of Human Induced Pluripotent Stem Cells Is Connected to the Reprogramming Process and Is Normalized During In Vitro Culture.

    PubMed

    Tesarova, Lenka; Simara, Pavel; Stejskal, Stanislav; Koutna, Irena

    2016-01-01

    The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications. PMID:27336948

  6. Cryopreserved hepatic progenitor cells derived from human embryonic stem cells can arrest progression of liver fibrosis in rats.

    PubMed

    Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra

    2016-10-01

    Hepatocytes generated from human embryonic stem cells (hESCs) are considered to be an excellent candidate for restoring the liver function deficiencies. We have earlier standardized a three-step differentiation protocol to generate functional hepatocyte-like cells (HLCs) from hESCs, which expressed the major hepatic markers. We have also found that the HLCs remain stable and functional even after extended period of in vitro culture and cryopreservation. In the present study, we have aimed to investigate the therapeutic potential of cryopreserved-thawed hESC-derived hepatic progenitor cells following transplantation in carbon tetrachloride-induced fibrotic rat livers. Significant therapeutic effects, including improved hepatic histology and normal serum biochemistry of hepatic enzymes along with increased survival rate, were observed in the cell transplanted rats. This result is an encouraging indication to develop methods for clinical application of hESC-derived hepatic lineage cells.

  7. The orphan nuclear receptor Nur77 regulates decidual prolactin expression in human endometrial stromal cells

    SciTech Connect

    Jiang, Yue; Hu, Yali; Zhao, Jing; Zhen, Xin; Yan, Guijun; Sun, Haixiang

    2011-01-14

    Research highlights: {yields} Decidually produced PRL plays a key role during pregnancy. {yields} Overexpression of Nur77 increased PRL mRNA expression and enhanced decidual PRL promoter activity. {yields} Knockdown of Nur77 decreased decidual PRL secretion induced by 8-Br-cAMP and MPA. {yields} Nur77 is a novel transcription factor that plays an active role in decidual prolactin expression. -- Abstract: Prolactin (PRL) is synthesized and released by several extrapituitary tissues, including decidualized stromal cells. Despite the important role of decidual PRL during pregnancy, little is understood about the factors involved in the proper regulation of decidual PRL expression. Here we present evidence that the transcription factor Nur77 plays an active role in decidual prolactin expression in human endometrial stromal cells (hESCs). Nur77 mRNA expression in hESCs was significantly increased after decidualization stimulated by 8-Br-cAMP and medroxyprogesterone acetate (MPA). Adenovirus-mediated overexpression of Nur77 in hESCs markedly increased PRL mRNA expression and enhanced decidual PRL promoter (dPRL/-332Luc) activity in a concentration-dependent manner. Furthermore, knockdown of Nur77 in hESCs significantly decreased decidual PRL promoter activation and substantially attenuated PRL mRNA expression and PRL secretion (P < 0.01) induced by 8-Br-cAMP and MPA. These results demonstrate that Nur77 is a novel transcription factor that contributes significantly to the regulation of prolactin gene expression in human endometrial stromal cells.

  8. The race is on: human embryonic stem cell research goes global.

    PubMed

    DeRouen, Mindy C; McCormick, Jennifer B; Owen-Smith, Jason; Scott, Christopher Thomas

    2012-12-01

    More nations are joining the human embryonic stem cell (hESC) "race" by aggressively publishing in the peer-reviewed journals. Here we present data on the international use and distribution of hESC using a dataset taken from the primary research literature. We extracted these papers from a comprehensive dataset of articles using hESC and human induced pluripotent stem cells (hiPSC). We find that the rate of publication by US-based authors is slowing in comparison to international labs, and then declines over the final year of the period 2008-2010. Non-US authors published more frequently and at a significantly higher rate, significantly increasing the number of their papers. In addition, international labs use a more diverse set of hESC lines and Obama-era additions are used more in non-US locations. Even considering the flood of new lines in the US and abroad, we see that researchers continue to rely on a few lines derived before the turn of the century. These data suggest "embargo" effects from restrictive policies on the US stem cell field. Over time, non-US labs have freely used lines on the US registries, while federally funded US scientists have been limited to using those lines approved by the NIH.

  9. Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila.

    PubMed Central

    Jablonski, P E; DiMarco, A A; Bobik, T A; Cabell, M C; Ferry, J G

    1990-01-01

    The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis. More than 100 mutually exclusive spots were present in acetate- and methanol-grown cells. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells. Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate- and methanol-grown Methanosarcina thermophila. Levels of formyltransferase in either acetate- or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum. All other enzyme activities were significantly lower in acetate- and methanol-grown Methanosarcina thermophila. Images FIG. 1 FIG. 2 PMID:2307649

  10. Use of a feline respiratory epithelial cell culture system grown at the air-liquid interface to characterize the innate immune response following feline herpesvirus 1 infection.

    PubMed

    Nelli, Rahul K; Maes, Roger; Kiupel, Matti; Hussey, Gisela Soboll

    2016-03-01

    Infection with feline herpesvirus-1 (FHV-1) accounts for 50% of viral upper respiratory diseases in domestic cats and is a significant cause of ocular diseases. Despite the clinical significance and high prevalence of FHV-1 infection, currently available vaccines cannot completely protect cats from infection and lifelong latency. FHV-1 infects via the mucous membranes and replicates in respiratory epithelial cells, but very little is known about the early innate immunity at this site. To address questions about immunity to FHV-1, feline respiratory epithelial cells cultured at air-liquid interface (ALI-FRECs) were established by collecting respiratory tracts from 6 healthy cats after euthanasia. Cells were isolated, cultured and characterized histologically and immunologically before infection with FHV-1. The expression of Toll-like receptors (TLRs), cytokine and chemokine responses were measured by real time PCR. ALI-FRECs morphologically resembled the natural airways of cats with multilayered columnar epithelial cells and cilia. Immunological properties of the natural airways were maintained in ALI-FRECs, as evidenced by the expression of TLRs, cytokines, chemokines, interferons, beta-defensins, and other regulatory genes. Furthermore, ALI-FRECs were able to support infection and replication of FHV-1, as well as modulate transcriptional regulation of various immune genes in response to infection. IL-1β and TNFα were increased in ALI-FRECs by 24hpi, whereas expression levels of IFN-α and TLR9 were not increased until 36hpi. In contrast, TLR3, GM-CSF and TGF-1β expression was down-regulated at 36hpi. The data presented show the development of a system ideal for investigating the molecular pathogenesis and immunity of FHV-1 or other respiratory pathogens.

  11. Potent inhibitory effect of δ-tocopherol on prostate cancer cells cultured in vitro and grown as xenograft tumors in vivo.

    PubMed

    Huang, Huarong; He, Yan; Cui, Xiao-Xing; Goodin, Susan; Wang, Hong; Du, Zhi Yun; Li, Dongli; Zhang, Kun; Tony Kong, Ah-Ng; DiPaola, Robert S; Yang, Chung S; Conney, Allan H; Zheng, Xi

    2014-11-01

    In the present study, the effects of δ-tocopherol (δ-T) on growth and apoptosis of human prostate cancer cells were determined and compared with that of α-tocopherol (α-T), a commonly used form of vitamin E. Treatment of human prostate cancer cells with δ-T resulted in strong growth inhibition and apoptosis stimulation, while the effects of α-T were modest. The strong effects of δ-T on the cells were associated with suppression of androgen receptor (AR) activity and decreased level of prostate specific antigen (PSA) that is a downstream target of the AR signaling. In the in vivo study, we found that δ-T had a more potent inhibitory effect on the formation and growth of prostate xenograft tumors than that of α-T. Moreover, δ-T inhibited proliferation and stimulated apoptosis in the tumors. The present study identified δ-T as a better form of vitamin E than α-T for future clinical studies of prostate cancer prevention.

  12. Biological behaviour of human umbilical artery smooth muscle cell grown on nickel-free and nickel-containing stainless steel for stent implantation

    PubMed Central

    Li, Liming; An, Liwen; Zhou, Xiaohang; Pan, Shuang; Meng, Xin; Ren, Yibin; Yang, Ke; Guan, Yifu

    2016-01-01

    To evaluate the clinical potential of high nitrogen nickel-free austenitic stainless steel (HNNF SS), we have compared the cellular and molecular responses of human umbilical artery smooth muscle cells (HUASMCs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel). CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profiles of HUASMCs exposed to HNNF SS and 316L SS, respectively. CCK-8 analysis demonstrated that HUASMCs cultured on HNNF SS proliferated more slowly than those on 316L SS. Flow cytometric analysis revealed that HNNF SS could activate more cellular apoptosis. The qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were up-regulated on HNNF SS. Thus, HNNF SS could reduce the HUASMC proliferation in comparison to 316L SS. The findings furnish valuable information for developing new biomedical materials for stent implantation. PMID:26727026

  13. Biological behaviour of human umbilical artery smooth muscle cell grown on nickel-free and nickel-containing stainless steel for stent implantation

    NASA Astrophysics Data System (ADS)

    Li, Liming; An, Liwen; Zhou, Xiaohang; Pan, Shuang; Meng, Xin; Ren, Yibin; Yang, Ke; Guan, Yifu

    2016-01-01

    To evaluate the clinical potential of high nitrogen nickel-free austenitic stainless steel (HNNF SS), we have compared the cellular and molecular responses of human umbilical artery smooth muscle cells (HUASMCs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel). CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profiles of HUASMCs exposed to HNNF SS and 316L SS, respectively. CCK-8 analysis demonstrated that HUASMCs cultured on HNNF SS proliferated more slowly than those on 316L SS. Flow cytometric analysis revealed that HNNF SS could activate more cellular apoptosis. The qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were up-regulated on HNNF SS. Thus, HNNF SS could reduce the HUASMC proliferation in comparison to 316L SS. The findings furnish valuable information for developing new biomedical materials for stent implantation.

  14. Selective MicroRNA-Offset RNA Expression in Human Embryonic Stem Cells

    PubMed Central

    Juhila, Juuso; Holm, Frida; Weltner, Jere; Trokovic, Ras; Mikkola, Milla; Toivonen, Sanna; Balboa, Diego; Lampela, Riina; Icay, Katherine; Tuuri, Timo; Otonkoski, Timo; Wong, Garry; Hovatta, Outi

    2015-01-01

    Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs. PMID:25822230

  15. Alu Sequences in Undifferentiated Human Embryonic Stem Cells Display High Levels of A-to-I RNA Editing

    PubMed Central

    Osenberg, Sivan; Paz Yaacov, Nurit; Safran, Michal; Moshkovitz, Sharon; Shtrichman, Ronit; Sherf, Ofra; Jacob-Hirsch, Jasmine; Keshet, Gilmor; Amariglio, Ninette; Itskovitz-Eldor, Joseph; Rechavi, Gideon

    2010-01-01

    Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3′UTRs, 5′UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference,