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Sample records for human antibody-secreting cells

  1. Specialized proresolving mediators enhance human B cell differentiation to antibody secreting cells1

    PubMed Central

    Ramon, Sesquile; Gao, Fei; Serhan, Charles N.; Phipps, Richard P.

    2012-01-01

    The resolution of inflammation is an active and dynamic process critical in maintaining homeostasis. Newly identified lipid mediators have been recognized as key players during the resolution phase. These specialized proresolving mediators (SPM) constitute separate families that include lipoxins, resolvins, protectins and maresins each derived from essential polyunsaturated fatty acids. New results demonstrate that SPM regulate aspects of the immune response, including reduction of neutrophil infiltration, decreased T cell cytokine production and stimulation of macrophage phagocytic activity. The actions of SPM on B lymphocytes remain unknown. Our study shows for the first time that the novel SPM 17-hydroxydosahexaenoic acid (17-HDHA), resolvin D1 (RvD1) and protectin D1 (PD1) are present in the spleen. Interestingly, 17-HDHA, RvD1 but not PD1, strongly increase activated human B cell IgM and IgG production. Furthermore, increased antibody production by 17-HDHA is due to augmented B cell differentiation towards a CD27+CD38+ antibody-secreting cell phenotype. 17-HDHA did not affect proliferation and was non-toxic to cells. Increase of plasma cell differentiation and antibody production supports the involvement of SPM during the late stages of inflammation and pathogen clearance. The present study provides new evidence for SPM activity in the humoral response. These new findings highlight the potential applications of SPM as endogenous and non-toxic adjuvants, and as anti-inflammatory therapeutic molecules. PMID:22711890

  2. Low-dose Anti-thymocyte Globulin Inhibits Human B-cell Differentiation into Antibody-Secreting Cells.

    PubMed

    Klaus, Pascal; Heine, Guido; Rasche, Claudia; Worm, Margitta

    2015-07-01

    Anti-thymocyte globulin (ATG) is used in the treatment of acute organ rejection. We studied in vitro the effect of low-dose ATG on B-cell activation and differentiation to antibody-secreting cells, as this may have an effect on B cell-driven autoimmune diseases, such as pemphigus vulgaris. Immunoglobulin production was analysed in the supernatants of peripheral blood mononuclear cells (PBMC) and CD19+ B cells from healthy donors and from patients with different autoimmune diseases. B-cell proliferation, viability and differentiation were analysed using flow cytometry. Differentiation of B cells to immunoglobulin G (IgG) secreting cells was significantly reduced by ATG, but not by control unspecific IgG from non-immunized rabbits (rIgG). B-cell viability was not altered by sub-depleting concentrations of ATG. In contrast, B-cell proliferation was enhanced by ATG. When PBMC from patients with autoimmune diseases were studied, specific autoantibodies could be detected in 1 out of 10 patients. In this patient, who had pemphigus vulgaris, ATG not only decreased total IgG, but decreased also specific anti-desmoglein-3. In conclusion, these data suggest that ATG at low concentrations inhibits B-cell differentiation and function. PMID:25587881

  3. Antibody secreting cell assay for influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISPOT assay to enumerate B-cells producing antibodies specific to a given antigen, also known as an antibody secreting cell (ASC) assay, was adapted to detect B-cells specific for influenza A virus (IAV). The assay is performed ex vivo and enumerates ASC at a single cell level. A simple ASC det...

  4. Characterization of Humanized Antibodies Secreted by Aspergillus niger

    PubMed Central

    Ward, Michael; Lin, Cherry; Victoria, Doreen C.; Fox, Bryan P.; Fox, Judith A.; Wong, David L.; Meerman, Hendrik J.; Pucci, Jeff P.; Fong, Robin B.; Heng, Meng H.; Tsurushita, Naoya; Gieswein, Christine; Park, Minha; Wang, Huaming

    2004-01-01

    Two different humanized immunoglobulin G1(κ) antibodies and an Fab′ fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex6GlcNAc2 to Hex15GlcNAc2. An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function. PMID:15128505

  5. Suppression of allo-human leucocyte antigen (HLA) antibodies secreted by B memory cells in vitro: intravenous immunoglobulin (IVIg) versus a monoclonal anti-HLA-E IgG that mimics HLA-I reactivities of IVIg

    PubMed Central

    Zhu, D; Ravindranath, M H; Terasaki, P I; Miyazaki, T; Pham, T; Jucaud, V

    2014-01-01

    B memory cells remain in circulation and secrete alloantibodies without antigen exposure > 20 years after alloimmunization postpartum or by transplantation. These long-lived B cells are resistant to cytostatic drugs. Therapeutically, intravenous immunoglobulin (IVIg) is administered to reduce allo-human leucocyte antigen (HLA) antibodies pre- and post-transplantation, but the mechanism of reduction remains unclear. Recently, we reported that IVIg reacts with several HLA-I alleles and the HLA reactivity of IVIg is lost after its HLA-E reactivity is adsorbed out. Therefore, we have generated an anti-HLA-E monoclonal antibody that mimics the HLA-reactivity of IVIg to investigate whether this antibody suppresses IgG secretion, as does IVIg. B cells were purified from the blood of a woman in whose blood the B memory cells remained without antigen exposure > 20 years after postpartum alloimmunization. The B cells were stimulated with cytokines using a well-defined culture system. The anti-HLA-E monoclonal antibody (mAb) significantly suppressed the allo-HLA class-II IgG produced by the B cells, and that this suppression was far superior to that by IVIg. These findings were confirmed with HLA-I antibody secreted by the immortalized B cell line, developed from the blood of another alloimmunized woman. The binding affinity of the anti-HLA-E mAb for peptide sequences shared (i.e. shared epitopes) between HLA-E and other β2-microglobulin-free HLA heavy chains (open conformers) on the cell surface of B cells may act as a ligand and signal suppression of IgG production of activated B memory cells. We propose that anti-HLA-E monoclonal antibody may also be useful to suppress allo-HLA IgG production in vivo. PMID:24611451

  6. Age-related changes in the transcriptome of antibody-secreting cells

    PubMed Central

    Kurupati, Raj; Showe, Louise C.; Ertl, Hildegund C.J.

    2016-01-01

    We analyzed age-related defects in B cell populations from young and aged mice. Microarray analysis of bone marrow resident antibody secreting cells (ASCs) showed significant changes upon aging, affecting multiple genes, pathways and functions including those that play a role in immune regulation, humoral immune responses, chromatin structure and assembly, cell metabolism and the endoplasmic reticulum (ER) stress response. Further analysis showed upon aging defects in energy production through glucose catabolism with reduced oxidative phosphorylation. In addition aged B cells had increased levels of reactive oxygen-species (ROS), which was linked to enhanced expression of the co-inhibitor programmed cell death (PD)-1. PMID:26967249

  7. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

    PubMed Central

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs. PMID:25996084

  8. The third dimension of ELISPOTs: quantifying antibody secretion from individual plasma cells.

    PubMed

    Bromage, Erin; Stephens, Rebecca; Hassoun, Lama

    2009-07-31

    The enzyme-linked immunospot assay (ELISPOT) is a technique widely used to enumerate the number of immune cells secreting a specific protein, such as antibodies or cytokines. A limitation with the ELISPOT assay is that it can only be used to detect a single protein of interest. Recently, the ELISPOT technique has been modified to use fluorophores allowing multiple secreted proteins to be detected simultaneously. This technique has greatly enhanced the ability to identify cells secreting multiple proteins, but has not been used to its fullest potential. We wished to accurately quantify the expression of antigen-specific antibody from a single plasma cell and to determine whether plasma cells recovered from different locations had different secretion rates. To achieve this we analyzed fluorospot images quantitatively using Mira MX 7 UL Astronomy software, and coupled this data with a quantitative ELISA to determine secretion rates from individual cells. Using this technique we were able to determine that plasma cells recovered from the peripheral blood secreted the most antibody (1.667 ng/cell/12 h) while splenic antibody secreting cells the least (0.399 ng/cell/12 h). We were able to quantify a 150 fold difference in antibody secretion between cells, with most plasma cells divided into two groups, low secretors (<0.1 ng/cell) or high secretors (>2 ng/cell). We believe this technique will be particularly useful for examining the secretion ratio of two proteins secreted from an individual cell, allowing us to determine if secretion is fixed or variable. PMID:19465022

  9. Differential requirement for OBF-1 during antibody-secreting cell differentiation

    PubMed Central

    Corcoran, Lynn M.; Hasbold, Jhagvaral; Dietrich, Wendy; Hawkins, Edwin; Kallies, Axel; Nutt, Stephen L.; Tarlinton, David M.; Matthias, Patrick; Hodgkin, Philip D.

    2005-01-01

    Resting B cells can be cultured to induce antibody-secreting cell (ASC) differentiation in vitro. A quantitative analysis of cell behavior during such a culture allows the influences of different stimuli and gene products to be measured. The application of this analytical system revealed that the OBF-1 transcriptional coactivator, whose loss impairs antibody production in vivo, has two effects on ASC development. Although OBF-1 represses early T cell–dependent (TD) differentiation, it is also critical for the completion of the final stages of ASC development. Under these conditions, the loss of OBF-1 blocks the genetic program of ASC differentiation so that Blimp-1/prdm1 induction fails, and bcl-6, Pax5, and AID are not repressed as in control ASC. Retroviral complementation confirmed that OBF-1 was the critical entity. Surprisingly, when cells were cultured in lipopolysaccharide to mimic T cell–independent conditions, OBF-1–null B cells differentiated normally to ASC. In the OBF-1−/− ASC generated under either culture regimen, antibody production was normal or only modestly reduced, revealing that Ig genes are not directly dependent on OBF-1 for their expression. The differential requirement for OBF-1 in TD ASC generation was confirmed in vivo. These studies define a new regulatory role for OBF-1 in determining the cell-autonomous capacity of B cells to undergo terminal differentiation in response to different immunological signals. PMID:15867091

  10. Number of specific antibody-secreting cells in the peripheral blood among children with mycoplasma pneumonia.

    PubMed Central

    Iseki, M; Takahashi, T; Kimura, K; Yamashita, R; Sasaki, T

    1996-01-01

    Mycoplasma pneumoniae-specific antibody-secreting cells (ASCs) in the peripheral blood were enumerated with an enzyme-linked immunospot assay in 12 children with mycoplasma pneumonia. Those cells were detected in the acute phases and declined in number in the convalescent stage. The maximum numbers of M. pneumoniae-specific ASCs ranged from 0 to 478 for immunoglobulin G (IgG), 13 to 1,992 for IgM, and 0 to 53 for IgA per 106 peripheral blood mononuclear cells, whereas the total numbers (i.e., including both specific and nonspecific) of immunoglobulin-secreting cells (IgSCs) were as high as 4,000 for both IgG and IgM and 1,000 for IgA per 106 peripheral blood mononuclear cells. Such a great increase in the numbers of total IgSCs in comparison with that in M. pneumoniae-specific ASCs suggests that the majority of the IgSC increase in the course of mycoplasmal infection was nonspecific to M. pneumoniae. The serum level of M. pneumoniae antibody measured by enzyme-linked immunosorbent assay remained high in the convalescent phase, while the number of specific ASCs decreased. Whereas this observation may be explained by declined degeneration or consumption of the antibody in the convalescent phase, it may be suggestive of the source of M. pneumoniae antibody other than ASCs in the peripheral blood. PMID:8698511

  11. Antibody Secreting Cell Responses following Vaccination with Bivalent Oral Cholera Vaccine among Haitian Adults

    PubMed Central

    Charles, Richelle C.; Mayo-Smith, Leslie M.; Teng, Jessica E.; Xu, Peng; Kováč, Pavol; Ryan, Edward T.; Qadri, Firdausi; Franke, Molly F.; Ivers, Louise C.; Harris, Jason B.

    2016-01-01

    Background The bivalent whole-cell (BivWC) oral cholera vaccine (Shanchol) is effective in preventing cholera. However, evaluations of immune responses following vaccination with BivWC have been limited. To determine whether BivWC induces significant mucosal immune responses, we measured V. cholerae O1 antigen-specific antibody secreting cell (ASC) responses following vaccination. Methodology/Principal Findings We enrolled 24 Haitian adults in this study, and administered doses of oral BivWC vaccine 14 days apart (day 0 and day 14). We drew blood at baseline, and 7 days following each vaccine dose (day 7 and 21). Peripheral blood mononuclear cells (PBMCs) were isolated, and ASCs were enumerated using an ELISPOT assay. Significant increases in Ogawa (6.9 cells per million PBMCs) and Inaba (9.5 cells per million PBMCs) OSP-specific IgA ASCs were detected 7 days following the first dose (P < 0.001), but not the second dose. The magnitude of V. cholerae-specific ASC responses did not appear to be associated with recent exposure to cholera. ASC responses measured against the whole lipolysaccharide (LPS) antigen and the OSP moiety of LPS were equivalent, suggesting that all or nearly all of the LPS response targets the OSP moiety. Conclusions/Significance Immunization with the BivWC oral cholera vaccine induced ASC responses among a cohort of healthy adults in Haiti after a single dose. The second dose of vaccine resulted in minimal ASC responses over baseline, suggesting that the current dosing schedule may not be optimal for boosting mucosal immune responses to V. cholerae antigens for adults in a cholera-endemic area. PMID:27308825

  12. Lymphocyte receptors. I. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells.

    PubMed Central

    McConnell, I; Hurd, C M

    1976-01-01

    Immunoglobulin (Ig) bearing, antigen-binding and antibody-secreting cells were investigated for the presence of membrane receptors for the Fc part of IgG and for C3b on their surface. Fc and C3b receptors were detected on the surface of most Ig-bearing and of most antigen-binding cells. Fc receptors were also detected on IgM antibody-secreting cells but not on IgG-secreting cells. C3b receptors were not detected on any antibody-secreting cells. These receptors are either lost from the B cell surface as they differentiate into antibody-secreting cells or are blocked in vivo by immune complexes or C3b. PMID:800392

  13. CD19 regulation of human B cell responses. B cell proliferation and antibody secretion are inhibited or enhanced by ligation of the CD19 surface glycoprotein depending on the stimulating signal used.

    PubMed

    Callard, R E; Rigley, K P; Smith, S H; Thurstan, S; Shields, J G

    1992-05-15

    The regulation of human B cell proliferation and differentiation by the CD19 surface glycoprotein was investigated. As expected, proliferation induced by costimulation with anti-IgM plus IL-4 or IL-2, or with G28.8 antibody plus IL-4 was inhibited by antibody ligation of CD19. In contrast, proliferation of tonsillar B cells to mitogenic doses of PMA (5 ng/ml) or to EBV were enhanced, and proliferation of B cell lines to BCGF(low) was unaffected. Similarly, specific antibody responses by tonsillar B cells to influenza virus, and Ig secretion by the CESS lymphoblastoid cell line in response to IL-6 were inhibited, whereas polyclonal Ig production in response to EBV was enhanced. These results show that human B cell responses may be inhibited or enhanced by CD19 depending on the stimulating signal used. The difference in response to CD19 ligation did not depend on whether proliferation or differentiation was being measured, or whether stimulation was by surface Ig. In experiments using PMA as a T cell independent mitogen, it was found that ligation of CD19 inhibited proliferation of B cells costimulated with low doses of PMA plus G28.5 (CD40) antibody, but enhanced the response to higher (mitogenic) doses with or without costimulation with G28.5. The change from inhibition to enhancement occurred over a very small increase in PMA dose (0.5-1.0 ng/ml) that corresponded exactly to the lowest dose required for mitogenic activity. Finally, we showed that CD19 ligation inhibited the increase in surface expression of CD23, but not IgM, induced by IL-4, showing that CD19 ligation can have opposed effects on different responses to the same signal. Together our results suggest that CD19 activation of human B cells interacts with other signaling events to enhance or inhibit the subsequent response. PMID:1374445

  14. Gold nanoparticles regulate the blimp1/pax5 pathway and enhance antibody secretion in B-cells

    NASA Astrophysics Data System (ADS)

    Lee, Chia-Hui; Syu, Shih-Han; Chen, Yu-Shiun; Hussain, Saber M.; Aleksandrovich Onischuk, Andrei; Chen, Wen Liang; Huang, G. Steven

    2014-03-01

    Nanoparticles are potential threats to human health and the environment; however, their medical applications as drug carriers targeting cancer cells bring hope to contemporary cancer therapy. As a model drug carrier, gold nanoparticles (GNPs) have been investigated extensively for in vivo toxicity. The effect of GNPs on the immune system, however, has rarely been examined. Antibody-secreting cells were treated with GNPs with diameters ranging from 2 to 50 nm. The GNPs enhanced IgG secretion in a size-dependent manner, with a peak of efficacy at 10 nm. The immune-stimulatory effect reached a maximum at 12 h after treatment but returned to control levels 24 h after treatment. This enhancing effect was validated ex vivo using B-cells isolated from mouse spleen. Evidence from RT-PCR and western blot experiments indicates that GNP-treatment upregulated B-lymphocyte-induced maturation protein 1 (blimp1) and downregulated paired box 5 (pax5). Immunostaining for blimp1 and pax5 in B-cells confirmed that the GNPs stimulated IgG secretion through the blimp1/pax5 pathway. The immunization of mice using peptide-conjugated GNPs indicated that the GNPs were capable of enhancing humoral immunity in a size-dependent manner. This effect was consistent with the bio-distribution of the GNPs in mouse spleen. In conclusion, in vitro, ex vivo, and in vivo evidence supports our hypothesis that GNPs enhance humoral immunity in mouse. The effect on the immune system should be taken into account if nanoparticles are used as carriers for drug delivery. In addition to their toxicity, the immune-stimulatory activity of nanoparticles could play an important role in human health and could have an environmental impact.

  15. CD138 and CD31 Double-Positive Cells Comprise the Functional Antibody-Secreting Plasma Cell Compartment in Primate Bone Marrow

    PubMed Central

    Martinez-Murillo, Paola; Pramanik, Lotta; Sundling, Christopher; Hultenby, Kjell; Wretenberg, Per; Spångberg, Mats; Karlsson Hedestam, Gunilla B.

    2016-01-01

    Plasma cells (PCs) are defined as terminally differentiated B cells that secrete large amounts of immunoglobulin (Ig). PCs that reside in the bone marrow (BM) are responsible for maintaining long-term antibody (Ab) responses after infection and vaccination, while PCs present in the blood are generally short-lived. In rhesus macaques, a species frequently used for the evaluation of human vaccines, B cells resemble those found in humans. However, a detailed characterization of BM-resident rhesus PC phenotype and function is lacking. Here, we examined Ig secretion of distinct rhesus CD138+ populations by B cell ELISpot analysis to couple phenotype with function. We demonstrate that the CD20low/−CD138+CD31+ BM population was highly enriched for antibody-secreting cells with IgG being the predominant isotype (60%), followed by IgA (33%) and IgM (7%). Transmission electron microscopy analysis confirmed PC enrichment in the CD20low/−CD138+CD31+ population with cells containing nuclei with “spokes of a wheel” chromatin structure and prominent rough endoplasmic reticulum. This panel also stained human BM PCs and allowed a clear distinction between BM PCs and short-lived peripheral PCs, providing an improved strategy to isolate PCs from rhesus BM for further analysis. PMID:27446073

  16. Transcription factor Ets1, but not the closely related factor Ets2, inhibits antibody-secreting cell differentiation.

    PubMed

    John, Shinu; Russell, Lisa; Chin, Shu Shien; Luo, Wei; Oshima, Robert; Garrett-Sinha, Lee Ann

    2014-02-01

    B cell differentiation into antibody-secreting cells (ASCs) is a tightly regulated process under the control of multiple transcription factors. One such transcription factor, Ets1, blocks the transition of B cells to ASCs via two separate activities: (i) stimulating the expression of target genes that promote B cell identity and (ii) interfering with the functional activity of the transcription factor Blimp1. Ets1 is a member of a multigene family, several members of which are expressed within the B cell lineage, including the closely related protein Ets2. In this report, we demonstrate that Ets1, but not Ets2, can block ASC formation despite the fact that Ets1 and Ets2 bind to apparently identical DNA sequence motifs and are thought to regulate overlapping sets of target genes. The DNA binding domain of Ets1 is required, but not sufficient by itself, to block ASC formation. In addition, less conserved regions within the N terminus of Ets1 play an important role in inhibiting B cell differentiation. Differences between the N termini of Ets1 and Ets2, rather than differences in the DNA binding domains, determine whether the proteins are capable of blocking ASC formation or not. PMID:24277931

  17. Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells.

    PubMed

    Lam, Wing Y; Becker, Amy M; Kennerly, Krista M; Wong, Rachel; Curtis, Jonathan D; Llufrio, Elizabeth M; McCommis, Kyle S; Fahrmann, Johannes; Pizzato, Hannah A; Nunley, Ryan M; Lee, Jieun; Wolfgang, Michael J; Patti, Gary J; Finck, Brian N; Pearce, Erika L; Bhattacharya, Deepta

    2016-07-19

    Durable antibody production after vaccination or infection is mediated by long-lived plasma cells (LLPCs). Pathways that specifically allow LLPCs to persist remain unknown. Through bioenergetic profiling, we found that human and mouse LLPCs could robustly engage pyruvate-dependent respiration, whereas their short-lived counterparts could not. LLPCs took up more glucose than did short-lived plasma cells (SLPCs) in vivo, and this glucose was essential for the generation of pyruvate. Glucose was primarily used to glycosylate antibodies, but glycolysis could be promoted by stimuli such as low ATP levels and the resultant pyruvate used for respiration by LLPCs. Deletion of Mpc2, which encodes an essential component of the mitochondrial pyruvate carrier, led to a progressive loss of LLPCs and of vaccine-specific antibodies in vivo. Thus, glucose uptake and mitochondrial pyruvate import prevent bioenergetic crises and allow LLPCs to persist. Immunizations that maximize these plasma cell metabolic properties might thus provide enduring antibody-mediated immunity. PMID:27396958

  18. Factors involved in CLL pathogenesis and cell survival are disrupted by differentiation of CLL B-cells into antibody-secreting cells

    PubMed Central

    Ghamlouch, Hussein; Darwiche, Walaa; Hodroge, Ahmed; Ouled-Haddou, Hakim; Dupont, Sébastien; Singh, Amrathlal Rabbind; Guignant, Caroline; Trudel, Stéphanie; Royer, Bruno

    2015-01-01

    Recent research has shown that chronic lymphocytic leukemia (CLL) B-cells display a strong tendency to differentiate into antibody-secreting cells (ASCs) and thus may be amenable to differentiation therapy. However, the effect of this differentiation on factors associated with CLL pathogenesis has not been reported. In the present study, purified CLL B-cells were stimulated to differentiate into ASCs by phorbol myristate acetate or CpG oligodeoxynucleotide, in combination with CD40 ligand and cytokines in a two-step, seven-day culture system. We investigated (i) changes in the immunophenotypic, molecular, functional, morphological features associated with terminal differentiation into ASCs, (ii) the expression of factors involved in CLL pathogenesis, and (iii) the expression of pro- and anti-apoptotic proteins in the differentiated cells. Our results show that differentiated CLL B-cells are able to display the transcriptional program of ASCs. Differentiation leads to depletion of the malignant program and deregulation of the apoptosis/survival balance. Analysis of apoptosis and the cell cycle showed that differentiation is associated with low cell viability and a low rate of cell cycle entry. Our findings shed new light on the potential for differentiation therapy as a part of treatment strategies for CLL. PMID:26050196

  19. Oct2 enhances antibody-secreting cell differentiation through regulation of IL-5 receptor α chain expression on activated B cells

    PubMed Central

    Emslie, Dianne; D'Costa, Kathy; Hasbold, Jhagvaral; Metcalf, Donald; Takatsu, Kiyoshi; Hodgkin, Philip O.; Corcoran, Lynn M.

    2008-01-01

    Mice lacking a functional gene for the Oct2 transcriptional activator display several developmental and functional deficiencies in the B lymphocyte lineage. These include defective B cell receptor (BCR) and Toll-like receptor 4 signaling, an absence of B-1 and marginal zone populations, and globally reduced levels of serum immunoglobulin (Ig) in naive and immunized animals. Oct2 was originally identified through its ability to bind to regulatory regions in the Ig loci, but genetic evidence has not supported an essential role for Oct2 in the expression of Ig genes. We describe a new Oct2-mediated role in B cells. Oct2 augments the ability of activated B cells to differentiate to antibody-secreting plasma cells (ASCs) under T cell–dependent conditions through direct regulation of the gene encoding the α chain of the interleukin (IL) 5 receptor. Ectopic expression of IL-5Rα in oct2-deficient B cells largely restores their ability to differentiate to functional ASCs in vitro but does not correct other phenotypic defects in the mutants, such as the maturation and specialization of peripheral B cells, which must therefore rely on distinct Oct2 target genes. IL-5 augments ASC differentiation in vitro, and we show that IL-5 directly activates the plasma cell differentiation program by enhancing blimp1 expression. PMID:18250192

  20. TLR9 Ligand (CpG Oligodeoxynucleotide) Induces CLL B-Cells to Differentiate into CD20+ Antibody-Secreting Cells

    PubMed Central

    Ghamlouch, Hussein; Ouled-Haddou, Hakim; Guyart, Aude; Regnier, Aline; Trudel, Stéphanie; Claisse, Jean-François; Fuentes, Vincent; Royer, Bruno; Marolleau, Jean-Pierre; Gubler, Brigitte

    2014-01-01

    B-cell chronic lymphocytic leukemia (CLL) is the most frequent adult leukemia in the Western world. It is a heterogeneous disease characterized by clonal proliferation and the accumulation of CD5+ mature B lymphocytes. However, the normal counterpart from which the latter cells arise has not yet been identified. CD27 expression and gene expression profiling data suggest that CLL cells are related to memory B-cells. In vitro, memory B-cells differentiate into plasma cells when stimulated with CpG oligodeoxynucleotide (CpG). The objective of the present study was therefore to investigate the ability of CpG, in the context of CD40 ligation, to induce the differentiation of CLL B-cells into antibody-secreting cells (ASCs). CD20+CD38− CLL B-cells were stimulated with a combination of CpG, CD40 ligand and cytokines (CpG/CD40L/c) in a two-step, 7-day culture system. We found that the CpG/CD40L/c culture system prompted CLL B-cells to differentiate into CD19+CD20+CD27+CD38−ASCs. These cells secreted large amounts of IgM and had the same shape as plasma cells. However, only IgMs secreted by ASCs that had differentiated from unmutated CLL B-cells were poly/autoreactive. Class-switch recombination (CSR) to IgG and IgA was detected in cells expressing the activation-induced cytidine deaminase gene (AICDA). Although these ASCs expressed high levels of the transcription factors PRDM1 (BLIMP1), IRF4, and XBP1s, they did not downregulate expression of PAX5. Our results suggest that CLL B-cells can differentiate into ASCs, undergo CSR and produce poly/autoreactive antibodies. Furthermore, our findings may be relevant for (i) identifying the normal counterpart of CLL B-cells and (ii) developing novel treatment strategies in CLL. PMID:24982661

  1. TLR9 Ligand (CpG Oligodeoxynucleotide) Induces CLL B-Cells to Differentiate into CD20(+) Antibody-Secreting Cells.

    PubMed

    Ghamlouch, Hussein; Ouled-Haddou, Hakim; Guyart, Aude; Regnier, Aline; Trudel, Stéphanie; Claisse, Jean-François; Fuentes, Vincent; Royer, Bruno; Marolleau, Jean-Pierre; Gubler, Brigitte

    2014-01-01

    B-cell chronic lymphocytic leukemia (CLL) is the most frequent adult leukemia in the Western world. It is a heterogeneous disease characterized by clonal proliferation and the accumulation of CD5(+) mature B lymphocytes. However, the normal counterpart from which the latter cells arise has not yet been identified. CD27 expression and gene expression profiling data suggest that CLL cells are related to memory B-cells. In vitro, memory B-cells differentiate into plasma cells when stimulated with CpG oligodeoxynucleotide (CpG). The objective of the present study was therefore to investigate the ability of CpG, in the context of CD40 ligation, to induce the differentiation of CLL B-cells into antibody-secreting cells (ASCs). CD20(+)CD38(-) CLL B-cells were stimulated with a combination of CpG, CD40 ligand and cytokines (CpG/CD40L/c) in a two-step, 7-day culture system. We found that the CpG/CD40L/c culture system prompted CLL B-cells to differentiate into CD19(+)CD20(+)CD27(+)CD38(-)ASCs. These cells secreted large amounts of IgM and had the same shape as plasma cells. However, only IgMs secreted by ASCs that had differentiated from unmutated CLL B-cells were poly/autoreactive. Class-switch recombination (CSR) to IgG and IgA was detected in cells expressing the activation-induced cytidine deaminase gene (AICDA). Although these ASCs expressed high levels of the transcription factors PRDM1 (BLIMP1), IRF4, and XBP1s, they did not downregulate expression of PAX5. Our results suggest that CLL B-cells can differentiate into ASCs, undergo CSR and produce poly/autoreactive antibodies. Furthermore, our findings may be relevant for (i) identifying the normal counterpart of CLL B-cells and (ii) developing novel treatment strategies in CLL. PMID:24982661

  2. The pathogenic role of virus-specific antibody-secreting cells in the central nervous system of rats with different susceptibility to coronavirus-induced demyelinating encephalitis.

    PubMed Central

    Schwender, S; Imrich, H; Dörries, R

    1991-01-01

    The humoral immune response in the central nervous system (CNS) of susceptible Lewis (LE) rats and resistant Brown Norway (BN) rats was analysed after intracerebral infection with the murine coronavirus JHM (MHV4). The subclinical course of the infection in BN rats was characterized by an early rise of neutralizing antibodies in the cerebrospinal fluid (CSF) 7 days post-infection. At this time in LE rats, neutralizing antibodies were not detectable in the CSF and the animals developed neurological signs of infection. Subsequently, LE rats recovered from disease. This process was accompanied by increasing titres of virus-neutralizing antibodies. Within the CNS parenchyma of both rat strains, equivalent numbers of IgM-secreting cells were detected. However, in BN rats, virus-specific IgG secreting cells appeared earlier and in higher numbers. Moreover, based on the size of zones of antibody secreted by single cells in the Spot-ELISA assay, it appeared that cells from BN rats secreted IgG antibody of higher affinity. These data suggest that early maturation of antiviral antibody responses in the resistant BN rat probably restricts the spread of viral infection to small foci within the CNS, resulting in a subclinical level of primary demyelination. In contrast, the absence of neutralizing antibodies in the susceptible LE rats favours spread of the virus throughout the CNS, resulting finally in severe neurological disease. Images Figure 1 Figure 2 Figure 3 PMID:1663078

  3. The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells

    PubMed Central

    Jurkin, Jennifer; Henkel, Theresa; Nielsen, Anne Færch; Minnich, Martina; Popow, Johannes; Kaufmann, Therese; Heindl, Katrin; Hoffmann, Thomas; Busslinger, Meinrad; Martinez, Javier

    2014-01-01

    The unfolded protein response (UPR) is a conserved stress-signaling pathway activated after accumulation of unfolded proteins within the endoplasmic reticulum (ER). Active UPR signaling leads to unconventional, enzymatic splicing of XBP1 mRNA enabling expression of the transcription factor XBP1s to control ER homeostasis. While IRE1 has been identified as the endoribonuclease required for cleavage of this mRNA, the corresponding ligase in mammalian cells has remained elusive. Here, we report that RTCB, the catalytic subunit of the tRNA ligase complex, and its co-factor archease mediate XBP1 mRNA splicing both in vitro and in vivo. Depletion of RTCB in plasma cells of Rtcbfl/fl Cd23-Cre mice prevents XBP1s expression, which normally is strongly induced during plasma cell development. RTCB-depleted plasma cells show reduced and disorganized ER structures as well as severe defects in antibody secretion. Targeting RTCB and/or archease thus represents a promising strategy for the treatment of a growing number of diseases associated with elevated expression of XBP1s. PMID:25378478

  4. Adjuvanted Intranasal Norwalk Virus-Like Particle Vaccine Elicits Antibodies and Antibody-Secreting Cells That Express Homing Receptors for Mucosal and Peripheral Lymphoid Tissues

    PubMed Central

    El-Kamary, Samer S.; Pasetti, Marcela F.; Mendelman, Paul M.; Frey, Sharon E.; Bernstein, David I.; Treanor, John J.; Ferreira, Jennifer; Chen, Wilbur H.; Sublett, Richard; Richardson, Charles; Bargatze, Robert F.; Sztein, Marcelo B.; Tacket, Carol O.

    2010-01-01

    Background. Noroviruses cause significant morbidity and mortality from acute gastroenteritis in all age groups worldwide. Methods.We conducted 2 phase 1 double-blind, controlled studies of a virus-like particle (VLP) vaccine derived from norovirus GI.1 genotype adjuvanted with monophosphoryl lipid A (MPL) and the mucoadherent chitosan. Healthy subjects 18–49 years of age were randomized to 2 doses of intranasal Norwalk VLP vaccine or controls 21 days apart. Study 1 evaluated 5-, 15-, and 50-μg dosages of Norwalk antigen, and study 2 evaluated 50-and 100-μg dosages. Volunteers recorded symptoms for 7 days after dosing, and safety was followed up for 180 days. Blood samples were collected for serological profile, antibody secreting cells (ASCs), and analysis of ASC homing receptors. Results. The most common symptoms were nasal stuffiness, discharge, and sneezing. No vaccine-related serious adverse events occurred. Norwalk VLP-specific immunoglobulin G and immunoglobulin A antibodies increased 4.8-and 9.1-fold, respectively, for the 100-μg dosage level. All subjects tested who received the 50-or 100-μg vaccine dose developed immunoglobulin A ASCs. These cells expressed molecules associated with homing to mucosal and peripheral lymphoid tissues. Conclusions. The intranasal monovalent adjuvanted Norwalk VLP vaccine was well tolerated and highly immunogenic and is a candidate for additional study. Trial Registration. ClinicalTrials.gov identifier: NCT00806962. PMID:20979455

  5. Total and Envelope Protein-Specific Antibody-Secreting Cell Response in Pediatric Dengue Is Highly Modulated by Age and Subsequent Infections

    PubMed Central

    Toro, Jessica F.; Salgado, Doris M.; Vega, Rocío; Rodríguez, Jairo A.; Rodríguez, Luz-Stella; Angel, Juana; Franco, Manuel A.; Greenberg, Harry B.; Narváez, Carlos F.

    2016-01-01

    The response of antibody-secreting cells (ASC) induced by dengue has only recently started to be characterized. We propose that young age and previous infections could be simple factors that affect this response. Here, we evaluated the primary and secondary responses of circulating ASC in infants (6–12 months old) and children (1–14 years old) infected with dengue showing different degrees of clinical severity. The ASC response was delayed and of lower magnitude in infants, compared with older children. In primary infection (PI), the total and envelope (E) protein-specific IgM ASC were dominant in infants but not in children, and a negative correlation was found between age and the number of IgM ASC (rho = −0.59, P = 0.03). However, infants with plasma dengue-specific IgG detectable in the acute phase developed an intense ASC response largely dominated by IgG and comparable to that of children with secondary infection (SI). IgM and IgG produced by ASC circulating in PI or SI were highly cross-reactive among the four serotypes. Dengue infection caused the disturbance of B cell subsets, particularly a decrease in the relative frequency of naïve B cells. Higher frequencies of total and E protein-specific IgM ASC in the infants and IgG in the children were associated with clinically severe forms of infection. Therefore, the ASC response induced by dengue is highly influenced by the age at which infection occurs and previous immune status, and its magnitude is a relevant element in the clinical outcome. These results are important in the search for correlates of protection and for determining the ideal age for vaccinating against dengue. PMID:27560782

  6. Total and Envelope Protein-Specific Antibody-Secreting Cell Response in Pediatric Dengue Is Highly Modulated by Age and Subsequent Infections.

    PubMed

    Toro, Jessica F; Salgado, Doris M; Vega, Rocío; Rodríguez, Jairo A; Rodríguez, Luz-Stella; Angel, Juana; Franco, Manuel A; Greenberg, Harry B; Narváez, Carlos F

    2016-01-01

    The response of antibody-secreting cells (ASC) induced by dengue has only recently started to be characterized. We propose that young age and previous infections could be simple factors that affect this response. Here, we evaluated the primary and secondary responses of circulating ASC in infants (6-12 months old) and children (1-14 years old) infected with dengue showing different degrees of clinical severity. The ASC response was delayed and of lower magnitude in infants, compared with older children. In primary infection (PI), the total and envelope (E) protein-specific IgM ASC were dominant in infants but not in children, and a negative correlation was found between age and the number of IgM ASC (rho = -0.59, P = 0.03). However, infants with plasma dengue-specific IgG detectable in the acute phase developed an intense ASC response largely dominated by IgG and comparable to that of children with secondary infection (SI). IgM and IgG produced by ASC circulating in PI or SI were highly cross-reactive among the four serotypes. Dengue infection caused the disturbance of B cell subsets, particularly a decrease in the relative frequency of naïve B cells. Higher frequencies of total and E protein-specific IgM ASC in the infants and IgG in the children were associated with clinically severe forms of infection. Therefore, the ASC response induced by dengue is highly influenced by the age at which infection occurs and previous immune status, and its magnitude is a relevant element in the clinical outcome. These results are important in the search for correlates of protection and for determining the ideal age for vaccinating against dengue. PMID:27560782

  7. The Distribution of SIgA and IgG Antibody-Secreting Cells in the Small Intestine of Bactrian Camels (Camelus bactrianus) of Different Ages

    PubMed Central

    Zhang, Wang-Dong; Wang, Wen-Hui; Jia, Shuai

    2016-01-01

    Secretory immunoglobulin A (SIgA) and immunoglobulin G (IgG) antibody-secreting cells (ASCs) are two important cell types in the mucosal immune system. This study aimed to explore the distribution of these ASC populations in the small intestine of Bactrian camels of different ages. Twenty-four Alashan Bactrian camels were divided into the following four age groups: young (1–2 years), pubertal (3–5 years), middle-aged (6–16 years) and old (17–20 years). SIgA and IgG ASCs in the intestinal mucosa lamina propria (LP) were observed and analyzed using immunohistochemcal techniques. The results from all age groups show that both SIgA and IgG ASCs were diffusely distributed in the intestinal LP, and some cells aggregated around the crypts. Moreover, the densities of the two ASC populations gradually increased from the duodenum to the jejunum and then decreased in the ileum. Meanwhile, there were more SIgA ASCs than IgG ASCs in the duodenum, jejunum, and ileum, and these differences were significant in the young and pubertal groups (P<0.05). In addition, the SIgA and IgG ASC densities increased from the young to the pubertal period, peaked at puberty, and then gradually decreased with age. The results demonstrate that the SIgA and IgG ASC distributions help to form two immunoglobulin barriers in the intestinal mucosa to provide full protection, helping to maintain homeostasis. These findings also underscore the importance of researching the development and degeneration of intestinal mucosal immunity in Bactrian camels. PMID:27249417

  8. The Distribution of SIgA and IgG Antibody-Secreting Cells in the Small Intestine of Bactrian Camels (Camelus bactrianus) of Different Ages.

    PubMed

    Zhang, Wang-Dong; Wang, Wen-Hui; Jia, Shuai

    2016-01-01

    Secretory immunoglobulin A (SIgA) and immunoglobulin G (IgG) antibody-secreting cells (ASCs) are two important cell types in the mucosal immune system. This study aimed to explore the distribution of these ASC populations in the small intestine of Bactrian camels of different ages. Twenty-four Alashan Bactrian camels were divided into the following four age groups: young (1-2 years), pubertal (3-5 years), middle-aged (6-16 years) and old (17-20 years). SIgA and IgG ASCs in the intestinal mucosa lamina propria (LP) were observed and analyzed using immunohistochemcal techniques. The results from all age groups show that both SIgA and IgG ASCs were diffusely distributed in the intestinal LP, and some cells aggregated around the crypts. Moreover, the densities of the two ASC populations gradually increased from the duodenum to the jejunum and then decreased in the ileum. Meanwhile, there were more SIgA ASCs than IgG ASCs in the duodenum, jejunum, and ileum, and these differences were significant in the young and pubertal groups (P<0.05). In addition, the SIgA and IgG ASC densities increased from the young to the pubertal period, peaked at puberty, and then gradually decreased with age. The results demonstrate that the SIgA and IgG ASC distributions help to form two immunoglobulin barriers in the intestinal mucosa to provide full protection, helping to maintain homeostasis. These findings also underscore the importance of researching the development and degeneration of intestinal mucosal immunity in Bactrian camels. PMID:27249417

  9. Antibody-secreting cell responses after Vibrio cholerae O1 infection and oral cholera vaccination in adults in Bangladesh.

    PubMed

    Rahman, Atiqur; Rashu, Rasheduzzaman; Bhuiyan, Taufiqur Rahman; Chowdhury, Fahima; Khan, Ashraful Islam; Islam, Kamrul; LaRocque, Regina C; Ryan, Edward T; Wrammert, Jens; Calderwood, Stephen B; Qadri, Firdausi; Harris, Jason B

    2013-10-01

    Infection with Vibrio cholerae and oral cholera vaccines (OCVs) induce transient circulating plasmablast responses that peak within approximately 7 days after infection or vaccination. We previously demonstrated that plasmablast responses strongly correlate with subsequent levels of V. cholerae-specific duodenal antibodies up to 6 months after V. cholerae infection. Hence, plasmablast responses provide an early window into the immunologic memory at the mucosal surface. In this study, we characterized plasmablast responses following V. cholerae infection using a flow cytometrically defined population and compared V. cholerae-specific responses in adult patients with V. cholerae O1 infection and vaccinees who received the OCV Dukoral (Crucell Vaccines Canada). Among flow cytometrically sorted populations of gut-homing plasmablasts, almost 50% of the cells recognized either cholera toxin B subunit (CtxB) or V. cholerae O1 lipopolysaccharide (LPS). Using a traditional enzyme-linked immunosorbent spot assay (ELISPOT), we found that infection with V. cholerae O1 and OCVs induce similar responses to the protein antigen CtxB, but responses to LPS were diminished after OCV compared to those after natural V. cholerae infection. A second dose of OCV on day 14 failed to boost circulating V. cholerae-specific plasmablast responses in Bangladeshi adults. Our results differ from those in studies from areas where cholera is not endemic, in which a second vaccination on day 14 significantly boosts plasmablast responses. Given these results, it is likely that the optimal boosting strategies for OCVs differ significantly between areas where V. cholerae infection is endemic and those where it is not. PMID:23945156

  10. Monoclonal antibodies against goldfish (Carassius auratus) immunoglobulin: application to the quantification of immunoglobulin and antibody-secreting cells by ELISPOT and seric immunoglobulin and antibody levels by ELISA in carp (Cyprinus carpio).

    PubMed

    Siwicki, A K; Vergnet, C; Charlemagne, J; Dunier, M

    1994-01-01

    Monoclonal antibodies (mAbs) raised against heavy and light chains of goldfish immunoglobulin (Ig) were characterized by a Western blot technique. A complete cross-reactivity was observed between carp and goldfish Ig. These mAbs were used for the quantification of carp Ig and anti-Yersinia ruckeri antibodies by ELISA. An ELISPOT assay was also developed in carp to quantify Ig-secreting cells (ISC) and antibody-secreting cells (ASC). The number of ASC was maximum on day 18 post-vaccination and decreased to the basal level on day 28. The antibody levels in sera were maximum on day 18 and slowly decreased until day 28. PMID:7951348

  11. Enumeration of Gut-Homing β7-Positive, Pathogen-Specific Antibody-Secreting Cells in Whole Blood from Enterotoxigenic Escherichia coli- and Vibrio cholerae-Infected Patients, Determined Using an Enzyme-Linked Immunosorbent Spot Assay Technique.

    PubMed

    Bhuiyan, Taufiqur Rahman; Hoq, Mohammad Rubel; Nishat, Naoshin Sharmin; Al Mahbuba, Deena; Rashu, Rasheduzzaman; Islam, Kamrul; Hossain, Lazina; Dey, Ayan; Harris, Jason B; Ryan, Edward T; Calderwood, Stephen B; Svennerholm, Ann-Mari; Qadri, Firdausi

    2016-01-01

    Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are noninvasive mucosal pathogens that cause acute watery diarrhea in people in developing countries. Direct assessment of the mucosal immune responses to these pathogens is problematic. Surrogate markers of local mucosal responses in blood are increasingly being studied to determine the mucosal immune responses after infection. However, the volume of blood available in children and infants has limited this approach. We assessed whether an approach that first isolates β7-positive cells from a small volume of blood would allow measurement of the antigen-specific immune responses in patients with cholera and ETEC infection. β7 is a cell surface marker associated with mucosal homing. We isolated β7-expressing cells from blood on days 2, 7, and 30 and used an enzyme-linked immunosorbent spot (ELISPOT) assay to assess the gut-homing antibody-secreting cells (ASCs) specific to pathogen antigens. Patients with ETEC diarrhea showed a significant increase in toxin-specific gut-homing ASCs at day 7 compared to the levels at days 2 and 30 after onset of illness and to the levels in healthy controls. Similar elevations of responses to the ETEC colonization factors (CFs) CS6 and CFA/I were observed in patients infected with CS6- and CFA/I-positive ETEC strains. Antigen-specific gut-homing ASCs to the B subunit of cholera toxin and cholera-specific lipopolysaccharides (LPS) were also observed on day 7 after the onset of cholera using this approach. This study demonstrates that a simple ELISPOT assay can be used to study the mucosal immunity to specific antigens using a cell-sorting protocol to isolate mucosal homing cells, facilitating measurement of mucosal responses in children following infection or vaccination. PMID:26512047

  12. Cells involved in the immune response. XXXVI. The thymic antigen-specific suppressor cell in the immunized rabbit is a T cell with receptors for FcG and the antigen and it acts, via a secreted suppressor factor, directly on the immune splenic AFC B cell to inhibit antibody secretion.

    PubMed Central

    Talor, E; Jodouin, C A; Richter, M

    1988-01-01

    Following i.v. immunization of the normal outbred rabbit with sheep (SRBC) or horse (HRBC) erythrocytes, antigen-specific suppressor cells are generated in the thymus capable of inhibiting the generation of haemolytic plaques by the autologous or allogeneic splenic antibody-forming cells (AFC) in the plaque-forming cell (PFC) assay. These suppressor cells secrete an antigen-specific suppressor factor in short-term (4-24 hr) culture in vitro. The suppressor cells are not detected in the thymus prior to Day 4, exhibit peak activity between Days 5 and 11 post-immunization, and decline slowly thereafter. Suppressor cells can no longer be detected in the thymus by Day 60 postimmunization. Suppressor cells are not detected in any of the other lymphoid organs of the immunized rabbit nor in any lymphoid organ in the unimmunized rabbit. The thymic suppressor cell is a T cell with surface receptors for the antigen (SRBC or HRBC) and for FcG. On the other hand, the AFC B cells generated in the spleen of the immunized rabbit possess cell-surface receptors for only the antigen and not for FcG. Both the suppressor cells and the secreted suppressor factor act directly on the AFC B lymphocytes to inhibit the generation of antigen-specific haemolytic plaques in the PFC assay. PMID:2455684

  13. Anti-DNA autoantibody-producing hybridomas of normal human lymphoid cell origin.

    PubMed Central

    Cairns, E; Block, J; Bell, D A

    1984-01-01

    Fusion of human myeloma cell line GM 4672 and tonsillar lymphoid cells from a normal donor resulted in 13 primary hybridomas, which produced IgM anti-single-stranded DNA (ssDNA) antibodies, as determined in enzyme-linked immunosorbent assay. Nine of these primary hybridomas have been cloned and a total of 34 clones were obtained. Supernatants of these cloned hybridomas were tested for binding to ssDNA, native DNA, RNA, low molecular weight supernatant DNA, polydeoxyguanylate-polydeoxycitidylate, polydeoxyadenylate-thymidylate sodium salt, and cardiolipin. Supernatants from all clones but one showed polyspecificity when reacting with the antigens tested. That the clones were true hybridomas rather than transformed lymphoid cells was evidence by IgM anti-DNA antibody secretion, karyotype analysis, and HLA typing. These studies imply that immunoglobulin genes encoding for anti-DNA autoantibodies with a spectrum of nucleic acid specificities similar to systemic lupus erythematosus, exist among normal B lymphocytes. PMID:6470143

  14. Cutaneous lymphocyte antigen expression on human effector B cells depends on the site and on the nature of antigen encounter.

    PubMed

    Kantele, Anu; Savilahti, Erkki; Tiimonen, Heidi; Iikkanen, Katja; Autio, Soile; Kantele, Jussi M

    2003-12-01

    In contrast to T cells, information on skin-homing B cells expressing the cutaneous lymphocyte antigen (CLA) is sparse. CLA expression on human B cells was investigated among circulating immunoglobulin-secreting cells (ISC) and among antigen-specific antibody-secreting cells (ASC) elicited by parenteral, oral or rectal primary immunization, or by parenteral or oral secondary immunization with Salmonella typhi Ty21a. CLA expression was examined by combining cell sorting with an enzyme-linked immunospot assay. Among all ISC, the proportion of CLA(+) cells was 13-21%. Parenteral immunization induced antigen-specific ASC of which 13% were CLA(+), while oral and rectal immunizations were followed by only 1% of CLA(+) ASC (p<0.001). Oral re-immunization was followed by an up-regulation of CLA (34-48%) regardless of the route of priming. Parenteral re-immunization elicited ASC of which 9-14% were CLA(+). In conclusion, the expression of CLA on human effector B cells depends on the site of antigen encounter: intestinal stimulation elicits cells with no CLA, while parenteral encounter elicits significant numbers of CLA(+) cells. Even though primary antigen encounter in the intestine failed to stimulate CLA expression, up-regulation of CLA was found upon intestinal antigen re-encounter. These findings may be of relevance in the pathogenesis of some cutaneous disorders. PMID:14635035

  15. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated disruption of the CD40 ligand-induced activation of primary human B cells

    SciTech Connect

    Lu Haitian Crawford, Robert B. Kaplan, Barbara L.F. Kaminski, Norbert E.

    2011-09-15

    Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells. - Highlights: > In this study primary human and mouse B cell toxicity to TCDD was compared. > TCDD altered the expression of Blimp-1 and Pax5 in mouse but not human B cells. > TCDD markedly suppressed human B cell activation as characterized by CD80, CD86 and CD69 expression. > TCDD inhibited ERK, p38, and Akt phosphorylation in human B cells.

  16. The B cell repertoire of patients with rheumatoid arthritis. Frequencies and specificities of peripheral blood B cells reacting with human IgG, human collagens, a mycobacterial heat shock protein and other antigens.

    PubMed Central

    Rudolphi, U; Hohlbaum, A; Lang, B; Peter, H H; Melchers, I

    1993-01-01

    Using a potent in vitro limiting dilution culture system, we have activated human peripheral blood B cells to proliferate and to differentiate into antibody-secreting cells (ASC). Under these conditions 25-100% of B cells are clonally expanded and produce IgM, IgG or IgA. Culture supernatants were tested for antibodies binding to human IgG-Fc fragments (RF), the 65-kD heat shock protein of Mycobacterium bovis (hsp60), human collagens type I, II, IV, V, transferrin, lactoferrin, albumins, and gelatine. All blood samples contained precursors of ASC (p-ASC) able to produce IgM binding to these antigens in frequencies above 0.03% of B cells. Most interestingly, a significant difference exists between rheumatoid arthritis (RA) patients and controls, concerning the relative frequencies of p-ASC able to produce monospecific or multireactive RF. Whereas most p-ASC(RF) in RA patients are monospecific (mean ratio 3.7), most p-ASC(RF) in healthy control persons are cross-reactive with at least one of five other antigens tested (mean ratio 0.2). The data suggest a disease-specific expansion of p-ASC committed to the production of monospecific rheumatoid factors. PMID:8099856

  17. IgG and IgA with potential microbial-binding activity are expressed by normal human skin epidermal cells.

    PubMed

    Jiang, Dongyang; Ge, Jing; Liao, Qinyuan; Ma, Junfan; Liu, Yang; Huang, Jing; Wang, Chong; Xu, Weiyan; Zheng, Jie; Shao, Wenwei; Lee, Gregory; Qiu, Xiaoyan

    2015-01-01

    The innate immune system of the skin is thought to depend largely on a multi-layered mechanical barrier supplemented by epidermis-derived antimicrobial peptides. To date, there are no reports of antimicrobial antibody secretion by the epidermis. In this study, we report the expression of functional immunoglobulin G (IgG) and immunoglobulin A (IgA), previously thought to be only produced by B cells, in normal human epidermal cells and the human keratinocyte line HaCaT. While B cells express a fully diverse Ig, epidermal cell-expressed IgG or IgA showed one or two conservative VHDJH rearrangements in each individual. These unique VDJ rearrangements in epidermal cells were found neither in the B cell-derived Ig VDJ databases published by others nor in our positive controls. IgG and IgA from epidermal cells of the same individual had different VDJ rearrangement patterns. IgG was found primarily in prickle cells, and IgA was mainly detected in basal cells. Both epidermal cell-derived IgG and IgA showed potential antibody activity by binding pathogens like Staphylococcus aureus, the most common pathogenic skin bacteria, but the microbial-binding profile was different. Our data indicates that normal human epidermal cells spontaneously express IgG and IgA, and we speculate that these Igs participate in skin innate immunity. PMID:25625513

  18. Mathematical modeling provides kinetic details of the human immune response to vaccination

    PubMed Central

    Le, Dustin; Miller, Joseph D.; Ganusov, Vitaly V.

    2015-01-01

    With major advances in experimental techniques to track antigen-specific immune responses many basic questions on the kinetics of virus-specific immunity in humans remain unanswered. To gain insights into kinetics of T and B cell responses in human volunteers we combined mathematical models and experimental data from recent studies employing vaccines against yellow fever and smallpox. Yellow fever virus-specific CD8 T cell population expanded slowly with the average doubling time of 2 days peaking 2.5 weeks post immunization. Interestingly, we found that the peak of the yellow fever-specific CD8 T cell response was determined by the rate of T cell proliferation and not by the precursor frequency of antigen-specific cells as has been suggested in several studies in mice. We also found that while the frequency of virus-specific T cells increased slowly, the slow increase could still accurately explain clearance of yellow fever virus in the blood. Our additional mathematical model described well the kinetics of virus-specific antibody-secreting cell and antibody response to vaccinia virus in vaccinated individuals suggesting that most of antibodies in 3 months post immunization were derived from the population of circulating antibody-secreting cells. Taken together, our analysis provided novel insights into mechanisms by which live vaccines induce immunity to viral infections and highlighted challenges of applying methods of mathematical modeling to the current, state-of-the-art yet limited immunological data. PMID:25621280

  19. Carbon Nanotubes and Human Cells?

    ERIC Educational Resources Information Center

    King, G. Angela

    2005-01-01

    Single-walled carbon nanotubes that were chemically altered to be water soluble are shown to enter fibroblasts, T cells, and HL60 cells. Nanoparticles adversely affect immortalized HaCaT human keratinocyte cultures, indicating that they may enter cells.

  20. Amitosis in human adrenal cells.

    PubMed

    Magalhães, M C; Pignatelli, D; Magalhães, M M

    1991-04-01

    Adrenal pieces obtained from 3 female and 2 male patients showed morphological figures of amitosis in adrenal zona reticularis cells. Such aspects were observed in both normal and hyperactive adrenals. Nuclei appeared constricted, heavily stained, with coarse chromatin, sometimes scattered among cytoplasmic organelles, but never marginating in crescentic caps. Cleavage of the cells originated two halves with a nucleolus in each pole. Binucleated cells were also seen in zona reticularis. The meaning of amitosis in human adrenal is discussed. PMID:1802124

  1. SIV replication in human cells

    PubMed Central

    Sakuma, Ryuta; Takeuchi, Hiroaki

    2012-01-01

    Current human immunodeficiency virus type 1 pandemic is believed to originate from cross-species transmission of simian immunodeficiency virus (SIV) into human population. Such cross-species transmission, however, is not efficient in general, because viral replication is modulated by host cell factors, with the species-specificity of these factors affecting viral tropism. An understanding of those host cell factors that affect viral replication contributes to elucidation of the mechanism for determination of viral tropism. This review will focus an anti-viral effect of ApoB mRNA editing catalytic subunit, tripartite motif protein 5 alpha, and cyclophilins on SIV replication and provide insight into the mechanism of species-specific barriers against viral infection in human cells. It will then present our current understanding of the mechanism that may explain zoonotic transmission of retroviruses. PMID:22679440

  2. Human Adrenocortical Carcinoma Cell Lines

    PubMed Central

    Wang, Tao; Rainey, William E.

    2011-01-01

    Summary The human adrenal cortex secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids are produced from unique cell types located within the three distinct zones of the adrenal cortex. Disruption of adrenal steroid production results in a variety of diseases that can lead to hypertension, metabolic syndrome, infertility and androgen excess. The adrenal cortex is also a common site for the development of adenomas, and rarely the site for the development of carcinomas. The adenomas can lead to diseases associated with adrenal steroid excess, while the carcinomas are particularly aggressive and have a poor prognosis. In vitro cell culture models provide an important tool to examine molecular and cellular mechanisms controlling both the normal and pathologic function of the adrenal cortex. Herein we discuss the human adrenocortical cell lines and their use as model systems for adrenal studies. PMID:21924324

  3. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  4. Human stem cell ethics: beyond the embryo.

    PubMed

    Sugarman, Jeremy

    2008-06-01

    Human embryonic stem cell research has elicited powerful debates about the morality of destroying human embryos. However, there are important ethical issues related to stem cell research that are unrelated to embryo destruction. These include particular issues involving different types of cells used, the procurement of such cells, in vivo use of stem cells, intellectual property, and conflicts of interest. PMID:18522846

  5. Embryonic Stem Cell Patents and Human Dignity

    PubMed Central

    Resnik, David B.

    2009-01-01

    This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells. PMID:17922198

  6. [Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire].

    PubMed

    Guillot-Chene, P; Lebecque, S; Rigal, D

    2009-05-01

    Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs. PMID:19446667

  7. Anti-DNA antibody production by CD5+ and CD5- B cells of patients with systemic lupus erythematosus.

    PubMed Central

    Suzuki, N; Sakane, T; Engleman, E G

    1990-01-01

    Although the presence of anti-DNA antibody is a hallmark of systemic lupus erythematosus (SLE), neither the subsets of B cells that secrete anti-DNA antibody nor the stimuli responsible for the induction of anti-DNA secretion is known. In particular, the role of CD5+ B cells in human SLE, a distinct subpopulation of antibody-secreting cells shown previously to be a source of anti-DNA antibody in murine models of SLE, is unknown. To approach these questions, we developed a sensitive enzyme-linked immunospot (ELIspot) assay to measure spontaneous secretion of antibody to single-stranded (ss) DNA, double-stranded (ds) DNA, tetanus toxoid, and polyclonal immunoglobulin (Ig) by purified CD5+ and CD5- B cells of 15 SLE patients and 15 healthy control subjects. The B cells of only 1 of 15 healthy subjects secreted a significant level of anti-ssDNA antibody, and none secreted anti-dsDNA. By contrast, in the majority of SLE patients both CD5+ and CD5- B cells secreted IgG and/or IgM anti-ssDNA as well as anti-dsDNA antibody. Further analysis of the anti-ssDNA response revealed that the level of IgG and IgM anti-DNA antibody secretion by CD5- B cells correlated closely with the level of polyclonal Ig production by the same subpopulation (r = 0.81 and 0.70, respectively). In contrast, production of anti-DNA by CD5+ B cells occurred independently of polyclonal Ig production by both CD5+ and CD5- B cell subpopulations. These results suggest that in human SLE there exist two anti-DNA antibody-producing B cell subpopulations with distinct induction mechanisms: one (CD5+), which independently secretes anti-DNA, and another (CD5-), which produces anti-DNA as an apparent consequence of polyclonal B cell activation. PMID:1688569

  8. Neoplastic transformation of human cells

    NASA Technical Reports Server (NTRS)

    Goth-Goldstein, Regine

    1995-01-01

    The goal of this project was to gain a better understanding of the cellular mechanisms of cancer induction by ionizing radiation as a risk assessment for workers subjected to high LET irradiation such as that found in space. The following ions were used for irradiation: Iron, Argon, Neon, and Lanthanum. Two tests were performed: growth in low serum and growth in agar were used as indicators of cell transformation. The specific aims of this project were to: (1) compare the effectiveness of various ions on degree of transformation of a single dose of the same RBE; (2) determine if successive irradiations with the same ion (Ge 600 MeV/u) increases the degree of transformation; (3) test if clones with the greatest degree of transformation produce tumors in nude mice; and (4) construct a cell hybrid of a transformed and control (non-transformed) clone. The cells used for this work are human mammary epithelial cells with an extended lifespan and selected for growth in MEM + 10% serum.

  9. Rapid Proliferation and Differentiation of a Subset of Circulating IgM Memory B Cells to a CpG/Cytokine Stimulus In Vitro

    PubMed Central

    Vásquez, Camilo; Franco, Manuel A.; Angel, Juana

    2015-01-01

    Circulating human IgM expressing memory B cells have been incompletely characterized. Here, we compared the phenotype and in vitro functional response (capacity to proliferate and differentiate to antibody secreting cells) in response to CpG and a cytokine cocktail (IL-2, IL-6, and IL-10) of sorted naïve B cells, IgM memory B cells and isotype-switched circulating memory B cells. Compared to naïve B cells, IgM memory B cells had lower integrated mean fluorescence intensity (iMFI) of BAFF-R, CD38, CD73, and IL-21R, but higher iMFI of CD95, CD11c, TLR9, PD-1, and CD122. Compared to switched memory B cells, IgM memory B cells had higher iMFI of BAFF-R, PD-1, IL-21R, TLR9, and CD122, but lower iMFI of CD38, CD95, and CD73. Four days after receiving the CpG/cytokine cocktail, higher frequencies of IgM than switched memory B cells—and these in turn greater than naïve cells—proliferated and differentiated to antibody secreting cells. At this time point, a small percentage (median of 7.6%) of stimulated IgM memory B cells changed isotype to IgG. Thus, among the heterogeneous population of human circulating IgM memory B cells a subset is capable of a rapid functional response to a CpG/cytokine stimulus in vitro. PMID:26439739

  10. Cell motion predicts human epidermal stemness

    PubMed Central

    Toki, Fujio; Tate, Sota; Imai, Matome; Matsushita, Natsuki; Shiraishi, Ken; Sayama, Koji; Toki, Hiroshi; Higashiyama, Shigeki

    2015-01-01

    Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation. PMID:25897083

  11. Approaches to Study Human T Cell Development.

    PubMed

    Dolens, Anne-Catherine; Van de Walle, Inge; Taghon, Tom

    2016-01-01

    Not only is human T cell development characterized by unique changes in surface marker expression, but it also requires specific growth factors and conditions to mimic and study T cell development in vitro. In this chapter, we provide an overview of the specific aspects that need attention when performing T cell differentiation cultures with human progenitors. PMID:26294413

  12. Systemic foot-and-mouth disease vaccination in cattle promotes specific antibody secreting cells at the respiratory tract and triggers local anamnestic-compatible responses upon aerosol infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot and mouth disease (FMD) is a highly contagious viral disease affecting biungulate species. Commercial vaccines, formulated with inactivated whole FMD virus (FMDV) particles, are regularly used worldwide in regions recognized as free from the disease. Here, we studied the generation of antibody ...

  13. Endothelial cells derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  14. Symmetry breaking in human neuroblastoma cells.

    PubMed

    Izumi, Hideki; Kaneko, Yasuhiko

    2014-01-01

    Asymmetric cell division (ACD) is a characteristic of cancer stem cells, which exhibit high malignant potential. However, the cellular mechanisms that regulate symmetric (self-renewal) and asymmetric cell divisions are mostly unknown. Using human neuroblastoma cells, we found that the oncosuppressor protein tripartite motif containing 32 (TRIM32) positively regulates ACD. PMID:27308367

  15. Symmetry breaking in human neuroblastoma cells

    PubMed Central

    Izumi, Hideki; Kaneko, Yasuhiko

    2014-01-01

    Asymmetric cell division (ACD) is a characteristic of cancer stem cells, which exhibit high malignant potential. However, the cellular mechanisms that regulate symmetric (self-renewal) and asymmetric cell divisions are mostly unknown. Using human neuroblastoma cells, we found that the oncosuppressor protein tripartite motif containing 32 (TRIM32) positively regulates ACD. PMID:27308367

  16. Trophoblast lineage cells derived from human induced pluripotent stem cells

    SciTech Connect

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  17. Human B-1 cells take the stage

    PubMed Central

    Rothstein, Thomas L.; Griffin, Daniel O.; Holodick, Nichol E.; Quach, Tam D.; Kaku, Hiroaki

    2013-01-01

    B-1cells play critical roles in defending against microbial invasion and in housekeeping removal of cellular debris. B-1cells secrete natural antibody and manifest functions that influence T cell expansion and differentiation and in these and other ways differ from conventional B-2 cells. B-1-cells were originally studied in mice where they are easily distinguished from B-2cells, but their identity in the human system remained poorly defined for many years. Recently, functional criteria for human B-1cells were established on the basis of murine findings, and reverse engineering resulted in identification of the phenotypic profile, CD20+CD27+CD43+CD70−, for B-1cells found in both umbilical cord blood and adult peripheral blood. Human B-1cells may contribute to multiple disease states through production of autoantibody and stimulation/modulation of T cell activity. Human B-1cells could be a rich source of antibodies useful in treating diseases present in elderly populations where natural antibody protection may have eroded. Manipulation of human B-1cell numbers and/or activity may be a new avenue for altering T cell function and treating immune dyscrasias. PMID:23692567

  18. A Transcriptional Regulatory Switch Underlying B-Cell Terminal Differentiation and Its Disruption by Dioxin (S)

    EPA Science Inventory

    The terminal differentiation of B cells in lymphoid organs into antibody-secreting plasma cells upon antigen stimulation is a crucial step in the humoral immune response. The architecture of the B-cell transcriptional regulatory network consists of coupled mutually-repressive fee...

  19. Cell culture: Progenitor cells from human brain after death

    NASA Astrophysics Data System (ADS)

    Palmer, Theo D.; Schwartz, Philip H.; Taupin, Philippe; Kaspar, Brian; Stein, Stuart A.; Gage, Fred H.

    2001-05-01

    Culturing neural progenitor cells from the adult rodent brain has become routine and is also possible from human fetal tissue, but expansion of these cells from postnatal and adult human tissue, although preferred for ethical reasons, has encountered problems. Here we describe the isolation and successful propagation of neural progenitor cells from human postmortem tissues and surgical specimens. Although the relative therapeutic merits of adult and fetal progenitor cells still need to be assessed, our results may extend the application of these progenitor cells in the treatment of neurodegenerative diseases.

  20. Modulation of human humoral immune response through orally administered bovine colostrum.

    PubMed

    He, F; Tuomola, E; Arvilommi, H; Salminen, S

    2001-08-01

    Eighteen healthy volunteers were randomized into two treatment groups and consumed liquid prepackaged bovine colostrum whey and placebo for 7 days. On days 1, 3 and 5, an attenuated Salmonella typhi Ty21a oral vaccine was given to all subjects to mimic an enteropathogenic infection. The circulating antibody secreting cells and the expression of phagocytosis receptors of the subjects before and after oral immunization were measured with the ELISPOT assay and flow cytometry. All subjects responded well to the vaccine. No significant differences were observed in ELISPOT values for IgA, IgG, IgM, Fcgamma and CR receptor expression on neutrophils and monocytes between the two groups. There was a trend towards greater increase in specific IgA among the subjects receiving their vaccine with bovine colostrum. These results suggest that bovine colostrum may possess some potential to enhance human special immune responses. PMID:11549415

  1. HIV-1 propagates in human neuroblastoma cells.

    PubMed

    Shapshak, P; Sun, N C; Resnick, L; Thornthwaite, J T; Schiller, P; Yoshioka, M; Svenningsson, A; Tourtellotte, W W; Imagawa, D T

    1991-01-01

    A major question in the pathogenesis of AIDS encephalopathy and dementia is whether HIV-1 directly infects cells of the central nervous system (CNS). The propagation of HIV was attempted in six cell lines: three related and three unrelated to the nervous system. HIV was able to propagate in two human neuroblastoma cell lines and a lymphocytic cell line control but did not result in infections of African green monkey kidney cells, human cervix carcinoma cells, and one human brain astrocytoma cell line. Neuroblastoma cell lines infected with HIV showed peaks of reverse transcriptase activity at 10-14 days postinfection. After prolonged growth in cell cultures, one of the neuroblastoma cell lines showed multiphasic virus production, additional high peaks of reverse transcriptase activity, 20-fold greater than the first, lasting from 36 to 74 days and 110 to 140 days postinfection. The presence of HIV was confirmed by p24 antigen capture. The neuroblastoma cell lines had weak but detectable levels of CD4 immunoreactivity by immunoperoxidase and flow immunocytometric analysis. Although no T4-specific RNA sequences were detected by hybridization of Northern blots of total and poly A-selected RNA extracted from the two neuroblastoma cell lines by using a T4 specific complimentary DNA probe, monoclonal antibodies to the CD4 receptor blocked HIV infection in both neuroblastoma cell lines. Thus, the infection of neuroblastoma cells by HIV occurs in part by a CD4-dependent mechanism. Passaging the neuroblastoma cell lines weekly and bimonthly resulted in similar cell cycle-DNA content patterns for the more permissive cell line and with significant numbers of cells in the S phase. HIV-infected neuroblastoma cell lines provide an in vitro model for the evaluation of virus-host cell interactions and may be useful in addressing the issue of the persistence of HIV in the human CNS. PMID:1704060

  2. Human Pulmonary Endothelial Cells in Culture

    PubMed Central

    Johnson, Alice R.

    1980-01-01

    Endothelial cells were cultured from various different human vessels, including aortas, pulmonary, ovarian, and umbilical arteries, and pulmonary, ovarian, and umbilical veins. The cultured cells were identified as endothelial cells by the presence of Factor VIII antigen and antiotensin I converting enzyme (kininase II). They retained these markers for at least five passages in culture, and some cells had them for seven passages or more. Endothelial cells from the various vessels were compared with respect to their ability to metabolize angiotensins I and II and bradykinin. Cells from arteries had three to five times the angiotensin I converting enzyme activity as cells from veins. The activity of angiotensinase A (aspartyl aminopeptidase) had a similar distribution, and cells from arteries were consistently more active than cells from veins. Cultures of endothelial cells from pulmonary and umbilical vessels formed prostacyclin in response to mechanical stimulation. Media from cell monolayers that were subjected to a change of medium and gentle agitation inhibited aggregation of human platelets. This inhibitory activity was generated within 2-5 min, and it was not formed by cells that were treated with indomethacin or tranylcypromine. Addition of prostaglandin (PG)H2 to indomethacin-treated cells restored the ability to form the inhibitor, but cells treated with tranylcypromine were not responsive to PGH2. In experiments where [14C]arachidonic acid was added to the cells before stimulation, the major metabolite identified by thin-layer chromatography was 6-keto PGF1α. Thus, it appears that pulmonary endothelial cells, as well as umbilical cord cells, can form prostacyclin. In experiments comparing the ability of arterial and venous cells to form prostacyclin, arterial cells were more active than venous cells. These studies of cells from various human vessels suggest that the vascular origin of cultured endothelial cells determines how they metabolize vasoactive

  3. Hepatic Differentiation from Human Ips Cells Using M15 Cells.

    PubMed

    Umeda, Kahoko; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    Here, we describe a procedure of human iPS cells differentiation into the definitive endoderm, further into albumin-expressing and albumin-secreting hepatocyte, using M15, a mesonephros- derived cell line. Approximately 90 % of human iPS cells differentiated into SOX17-positive definitive endoderm then approximately 50 % of cells became albumin-positive cells, and secreted ALB protein. This M15 feeder system for endoderm and hepatic differentiation is a simple and efficient method, and useful for elucidating molecular mechanisms for hepatic fate decision, and could represent an attractive approach for a surrogate cell source for pharmaceutical studies. PMID:25417065

  4. Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

    PubMed Central

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L.; Wolf, Don; Mitalipov, Shoukhrat

    2013-01-01

    SUMMARY Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state. PMID:23683578

  5. Human neuroepithelial cells express NMDA receptors.

    PubMed

    Sharp, Christopher D; Fowler, M; Jackson, T H; Houghton, J; Warren, A; Nanda, A; Chandler, I; Cappell, B; Long, A; Minagar, A; Alexander, J S

    2003-11-13

    L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1) cerebral endothelial barrier and 2) cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells) have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR) expression via immunohistochemistry and murine neuroepithelial cell line (V1) were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease. PMID:14614784

  6. Vascular Potential of Human Pluripotent Stem Cells

    PubMed Central

    Iacobas, Ionela; Vats, Archana; Hirschi, Karen K.

    2010-01-01

    Cardiovascular disease is the number one cause of death and disability in the US. Understanding the biological activity of stem and progenitor cells, and their ability to contribute to the repair, regeneration and remodeling of the heart and blood vessels affected by pathologic processes is an essential part of the paradigm in enabling us to achieve a reduction in related deaths. Both human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources of cells for clinical cardiovascular therapies. Additional in vitro studies are needed, however, to understand their relative phenotypes and molecular regulation toward cardiovascular cell fates. Further studies in translational animal models are also needed to gain insights into the potential and function of both human ES- and iPS-derived cardiovascular cells, and enable translation from experimental and pre-clinical studies to human trials. PMID:20453170

  7. A Transcriptional Regulatory Switch Underlying B-Cell Terminal Differentiation and its Disruption by Dioxin

    EPA Science Inventory

    The terminal differentiation of B lymphocytes into antibody-secreting plasma cells upon antigen stimulation is a crucial step in the humoral immune response. The mutually-repressive interactions among three key regulatory transcription factors underlying B to plasma cell differe...

  8. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    NASA Technical Reports Server (NTRS)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  9. Unique multipotent cells in adult human mesenchymal cell populations

    PubMed Central

    Kuroda, Yasumasa; Kitada, Masaaki; Wakao, Shohei; Nishikawa, Kouki; Tanimura, Yukihiro; Makinoshima, Hideki; Goda, Makoto; Akashi, Hideo; Inutsuka, Ayumu; Niwa, Akira; Shigemoto, Taeko; Nabeshima, Yoko; Nakahata, Tatsutoshi; Nabeshima, Yo-ichi; Fujiyoshi, Yoshinori; Dezawa, Mari

    2010-01-01

    We found adult human stem cells that can generate, from a single cell, cells with the characteristics of the three germ layers. The cells are stress-tolerant and can be isolated from cultured skin fibroblasts or bone marrow stromal cells, or directly from bone marrow aspirates. These cells can self-renew; form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency; and can differentiate into endodermal, ectodermal, and mesodermal cells both in vitro and in vivo. When transplanted into immunodeficient mice by local or i.v. injection, the cells integrated into damaged skin, muscle, or liver and differentiated into cytokeratin 14-, dystrophin-, or albumin-positive cells in the respective tissues. Furthermore, they can be efficiently isolated as SSEA-3(+) cells. Unlike authentic ES cells, their proliferation activity is not very high and they do not form teratomas in immunodeficient mouse testes. Thus, nontumorigenic stem cells with the ability to generate the multiple cell types of the three germ layers can be obtained through easily accessible adult human mesenchymal cells without introducing exogenous genes. These unique cells will be beneficial for cell-based therapy and biomedical research. PMID:20421459

  10. Toxicity of diuron in human cancer cells.

    PubMed

    Huovinen, Marjo; Loikkanen, Jarkko; Naarala, Jonne; Vähäkangas, Kirsi

    2015-10-01

    Diuron is a substituted phenylurea used as a herbicide to control broadleaf and grass weeds and as a biocidal antifouling agent. Diuron is carcinogenic in rat urinary bladder and toxic to the reproductive system of oysters, sea urchins and lizards. The few studies carried out in human cells do not include the genotoxicity of diuron. We have investigated the toxicity of diuron in human breast adenocarcinoma (MCF-7) and human placental choriocarcinoma (BeWo) cells. The production of reactive oxygen species (ROS) was statistically significantly increased in both cell lines but only at the highest 200 μM concentration. Diuron clearly reduced the viability of BeWo, but not MCF-7 cells. The relative cell number was decreased in both cell lines indicative of inhibition of cell proliferation. In the Comet assay, diuron increased DNA fragmentation in MCF-7 but not in BeWo cells. The expressions of p53 protein, a marker for cell stress, and p21 protein, a transcriptional target of p53, were increased, but only in MCF-7 cells. In conclusion, our results suggest that diuron is cytotoxic and potentially genotoxic in a tissue-specific manner and that ROS play a role in its toxicity. Thus, exposure to diuron may exert harmful effects on fetal development and damage human health. PMID:26086120

  11. Benign Mesenchymal Stromal Cells in Human Sarcomas

    PubMed Central

    Morozov, Alexei; Downey, Robert J.; Healey, John; Moreira, Andre L.; Lou, Emil; Leung, Roland; Edgar, Mark; Singer, Samuel; LaQuaglia, Michael; Maki, Robert G.; Moore, Malcolm A.S.

    2010-01-01

    Purpose Recent evidence suggests that at least some sarcomas arise through aberrant differentiation of mesenchymal stromal cells (MSCs), but MSCs have never been isolated directly from human sarcoma specimens. Experimental Design We examined human sarcoma cell lines and primary adherent cultures derived from human sarcoma surgical samples for features of MSCs. We further characterized primary cultures as either benign or malignant by the presence of tumor-defining genetic lesions and tumor formation in immunocompromised mice. Results We show that a dedifferentiated liposarcoma cell line DDLS8817 demonstrates fat, bone and cartilage trilineage differentiation potential characteristic of MSCs. Primary sarcoma cultures have the morphology, surface immunophenotype and differentiation potential characteristic of MSCs. Surprisingly, many of these cultures are benign as they do not form tumors in mice and lack sarcoma-defining genetic lesions. Consistent with the recently proposed pericyte origin of MSCs in normal human tissues, sarcoma-derived benign MSCs express markers of pericytes and cooperate with endothelial cells in tube formation assays. In human sarcoma specimens, a subset of CD146-positive microvascular pericytes express CD105, an MSC marker, while malignant cells largely do not. In an in vitro co-culture model, sarcoma-derived benign MSCs as well as normal human pericytes markedly stimulate the growth of sarcoma cell lines. Conclusions Sarcoma-derived benign MSCs/pericytes represent a previously undescribed stromal cell type in sarcoma which may contribute to tumor formation. PMID:21138865

  12. Immortalisation of human urothelial cells.

    PubMed

    Petzoldt, J L; Leigh, I M; Duffy, P G; Sexton, C; Masters, J R

    1995-01-01

    A cell line derived from the urothelium lining the ureter of a 12-year-old girl was immortalised using a temperature-sensitive SV40 large T-antigen gene construct, and designated UROtsa. Following immortalisation, UROtsa cells expressed SV40 large T-antigen, but did not acquire characteristics of neoplastic transformation, including growth in soft agar or the development of tumours in nude mice. Metaphase spreads had a normal chromosomal appearance and number. UROtsa cells remained permissive for cell growth at 39 degrees C, indicating that they did not retain temperature sensitivity. UROtsa provides an in vitro model of "normal" urothelium. PMID:8788275

  13. Reprogramming of human somatic cells by bacteria.

    PubMed

    Ito, Naofumi; Ohta, Kunimasa

    2015-05-01

    In general, it had been believed that the cell fate restriction of terminally differentiated somatic cells was irreversible. In 1952, somatic cell nuclear transfer (SCNT) was introduced to study early embryonic development in frogs. So far, various mammalian species have been successfully cloned using the SCNT technique, though its efficiency is very low. Embryonic stem (ES) cells were the first pluripotent cells to be isolated from an embryo and have a powerful potential to differentiate into more than 260 types of cells. The generation of induced pluripotent stem (iPS) cells was a breakthrough in stem cell research, and the use of these iPS cells has solved problems such as low efficiency and cell fate restriction. These cells have since been used for clinical application, disease investigation, and drug selection. As it is widely accepted that the endosymbiosis of Archaea into eukaryotic ancestors resulted in the generation of eukaryotic cells, we examined whether bacterial infection could alter host cell fate. We previously showed that when human dermal fibroblast (HDF) cells were incorporated with lactic acid bacteria (LAB), the LAB-incorporated HDF cells formed clusters and expressed a subset of common pluripotent markers. Moreover, LAB-incorporated cell clusters could differentiate into cells derived from each of the three germinal layers both in vivo and in vitro, indicating successful reprogramming of host HDF cells by LAB. In the current review, we introduce the existing examples of cellular reprogramming by bacteria and discuss their nuclear reprogramming mechanisms. PMID:25866152

  14. The Human Natural Killer Cell Immune Synapse

    NASA Astrophysics Data System (ADS)

    Davis, Daniel M.; Chiu, Isaac; Fassett, Marlys; Cohen, George B.; Mandelboim, Ofer; Strominger, Jack L.

    1999-12-01

    Inhibitory killer Ig-like receptors (KIR) at the surface of natural killer (NK) cells induced clustering of HLA-C at the contacting surface of target cells. In this manner, inhibitory immune synapses were formed as human NK cells surveyed target cells. At target/NK cell synapses, HLA-C/KIR distributed into rings around central patches of intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, the opposite orientation to mature murine T cell-activating synapses. This organization of protein was stable for at least 20 min. Cells could support multiple synapses simultaneously, and clusters of HLA-C moved as NK cells crawled over target cells. Clustering required a divalent metal cation, explaining how metal chelators inhibit KIR function. Surprisingly, however, formation of inhibitory synapses was unaffected by ATP depletion and the cytoskeletal inhibitors, colchicine and cytochalsins B and D. Clearly, supramolecular organization within plasma membranes is critical for NK cell immunosurveillance.

  15. The fate of human Treg cells.

    PubMed

    Battaglia, Manuela; Roncarolo, Maria Grazia

    2009-06-19

    In this issue of Immunity, Miyara et al. (2009) demonstrate that FoxP3(+) cells in human peripheral blood are heterogeneous in function, and CD45RA expression defines their different stages of differentiation. PMID:19538927

  16. Germ cell quantitation in human testicular biopsy.

    PubMed

    Sinha Hikim, A P; Chakraborty, J; Jhunjhunwala, J S

    1985-01-01

    Quantitative analysis of human seminiferous epithelium was carried out using an improved method of glutaraldehyde and osmium fixation with plastic embedding. Part of each biopsy specimen was fixed in Bouin's fixative and embedded in paraffin for comparison. Epon embedded tissue had very little artifactual damage compared with paraffin embedded tissue sections. The germ cell to Sertoli cell ratios were determined by counting the various germ cells per "unit" tubular area. Data obtained by this method reflect a remarkable stability of Sertoli cell number and germ cell-Sertoli cell ratios both between biopsies from different individuals and between biopsies from right and left testes from the same individual. Agreement between the present results and those of earlier studies based on paraffin embedded testicular specimens supports the validity of this method of germ cell quantitation of human testicular biopsy samples. PMID:3927550

  17. REPLICATIVE POTENTIAL OF HUMAN NATURAL KILLER CELLS

    PubMed Central

    Fujisaki, Hiroyuki; Kakuda, Harumi; Imai, Chihaya; Mullighan, Charles G.; Campana, Dario

    2009-01-01

    The replicative potential of human CD56+ CD3− natural killer (NK) cells is unknown. We found that by exposing NK cells to the leukemic cell line K562 genetically modified to express 4-1BB ligand and interleukin 15 (K562-mb15-41BBL), they expanded for up to 30 population doublings, achieving numbers that ranged from 1.6 × 105 to 1.2 × 1011 percent (median, 5.9 × 106 percent; n = 7) of those originally seeded. However, NK cells eventually became unresponsive to stimulation and died. Their demise could be suppressed by enforcing the expression of the human telomerase reverse transcriptase (TERT) gene. TERT-overexpressing NK cells continued to proliferate in response to K562-mb15-41BBL stimulation for more than 1 year of culture, while maintaining a normal karyotype and genotype. Long-lived NK cells had high cytotoxicity against myeloid and T-lineage leukemic cells. They remained susceptible to genetic manipulation, becoming highly cytotoxic to B-lineage leukemic cells after expression of anti-CD19 signaling receptors. Thus, human NK cells have a replicative potential similar to that of T lymphocytes and their lifespan can be significantly prolonged by an increase in TERT activity. We suggest that the methods described here should have many applications in studies of NK cell biology and NK cell-based therapies. PMID:19344420

  18. Satellite cells in human skeletal muscle plasticity

    PubMed Central

    Snijders, Tim; Nederveen, Joshua P.; McKay, Bryon R.; Joanisse, Sophie; Verdijk, Lex B.; van Loon, Luc J. C.; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models. PMID:26557092

  19. Human progenitor cells for bone engineering applications.

    PubMed

    de Peppo, G M; Thomsen, P; Karlsson, C; Strehl, R; Lindahl, A; Hyllner, J

    2013-06-01

    In this report, the authors review the human skeleton and the increasing burden of bone deficiencies, the limitations encountered with the current treatments and the opportunities provided by the emerging field of cell-based bone engineering. Special emphasis is placed on different sources of human progenitor cells, as well as their pros and cons in relation to their utilization for the large-scale construction of functional bone-engineered substitutes for clinical applications. It is concluded that, human pluripotent stem cells represent a valuable source for the derivation of progenitor cells, which combine the advantages of both embryonic and adult stem cells, and indeed display high potential for the construction of functional substitutes for bone replacement therapies. PMID:23642054

  20. 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

  1. Mitochondria in human pluripotent stem cell apoptosis.

    PubMed

    TeSlaa, Tara; Setoguchi, Kiyoko; Teitell, Michael A

    2016-04-01

    Human pluripotent stem cells (hPSCs) have great potential in regenerative medicine because they can differentiate into any cell type in the body. Genome integrity is vital for human development and for high fidelity passage of genetic information across generations through the germ line. To ensure genome stability, hPSCs maintain a lower rate of mutation than somatic cells and undergo rapid apoptosis in response to DNA damage and additional cell stresses. Furthermore, cellular metabolism and the cell cycle are also differentially regulated between cells in pluripotent and differentiated states and can aid in protecting hPSCs against DNA damage and damaged cell propagation. Despite these safeguards, clinical use of hPSC derivatives could be compromised by tumorigenic potential and possible malignant transformation from failed to differentiate cells. Since hPSCs and mature cells differentially respond to cell stress, it may be possible to specifically target undifferentiated cells for rapid apoptosis in mixed cell populations to enable safer use of hPSC-differentiated cells in patients. PMID:26828436

  2. 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.

  3. Human Stem Cells for Craniomaxillofacial Reconstruction

    PubMed Central

    Kirkpatrick, William Niall Alexander; Cameron, Malcolm Gregor

    2014-01-01

    Human stem cell research represents an exceptional opportunity for regenerative medicine and the surgical reconstruction of the craniomaxillofacial complex. The correct architecture and function of the vastly diverse tissues of this important anatomical region are critical for life supportive processes, the delivery of senses, social interaction, and aesthetics. Craniomaxillofacial tissue loss is commonly associated with inflammatory responses of the surrounding tissue, significant scarring, disfigurement, and psychological sequelae as an inevitable consequence. The in vitro production of fully functional cells for skin, muscle, cartilage, bone, and neurovascular tissue formation from human stem cells, may one day provide novel materials for the reconstructive surgeon operating on patients with both hard and soft tissue deficit due to cancer, congenital disease, or trauma. However, the clinical translation of human stem cell technology, including the application of human pluripotent stem cells (hPSCs) in novel regenerative therapies, faces several hurdles that must be solved to permit safe and effective use in patients. The basic biology of hPSCs remains to be fully elucidated and concerns of tumorigenicity need to be addressed, prior to the development of cell transplantation treatments. Furthermore, functional comparison of in vitro generated tissue to their in vivo counterparts will be necessary for confirmation of maturity and suitability for application in reconstructive surgery. Here, we provide an overview of human stem cells in disease modeling, drug screening, and therapeutics, while also discussing the application of regenerative medicine for craniomaxillofacial tissue deficit and surgical reconstruction. PMID:24564584

  4. Growth of gold nanoparticles in human cells.

    PubMed

    Anshup, Anshup; Venkataraman, J Sai; Subramaniam, Chandramouli; Kumar, R Rajeev; Priya, Suma; Kumar, T R Santhosh; Omkumar, R V; John, Annie; Pradeep, T

    2005-12-01

    Gold nanoparticles of 20-100 nm diameter were synthesized within HEK-293 (human embryonic kidney), HeLa (human cervical cancer), SiHa (human cervical cancer), and SKNSH (human neuroblastoma) cells. Incubation of 1 mM tetrachloroaurate solution, prepared in phosphate buffered saline (PBS), pH 7.4, with human cells grown to approximately 80% confluency yielded systematic growth of nanoparticles over a period of 96 h. The cells, stained due to nanoparticle growth, were adherent to the bottom of the wells of the tissue culture plates, with their morphology preserved, indicating that the cell membrane was intact. Transmission electron microscopy of ultrathin sections showed the presence of nanoparticles within the cytoplasm and in the nucleus, the latter being much smaller in dimension. Scanning near field microscopic images confirmed the growth of large particles within the cytoplasm. Normal cells gave UV-visible signatures of higher intensity than the cancer cells. Differences in the cellular metabolism of cancer and noncancer cells were manifested, presumably in their ability to carry out the reduction process. PMID:16316080

  5. Bioinformatics construction of the human cell surfaceome

    PubMed Central

    da Cunha, J. P. C.; Galante, P. A. F.; de Souza, J. E.; de Souza, R. F.; Carvalho, P. M.; Ohara, D. T.; Moura, R. P.; Oba-Shinja, S. M.; Marie, S. K. N.; Silva, W. A.; Perez, R. O.; Stransky, B.; Pieprzyk, M.; Moore, J.; Caballero, O.; Gama-Rodrigues, J.; Habr-Gama, A.; Kuo, W. P.; Simpson, A. J.; Camargo, A. A.; Old, Lloyd J.; de Souza, S. J.

    2009-01-01

    Cell surface proteins are excellent targets for diagnostic and therapeutic interventions. By using bioinformatics tools, we generated a catalog of 3,702 transmembrane proteins located at the surface of human cells (human cell surfaceome). We explored the genetic diversity of the human cell surfaceome at different levels, including the distribution of polymorphisms, conservation among eukaryotic species, and patterns of gene expression. By integrating expression information from a variety of sources, we were able to identify surfaceome genes with a restricted expression in normal tissues and/or differential expression in tumors, important characteristics for putative tumor targets. A high-throughput and efficient quantitative real-time PCR approach was used to validate 593 surfaceome genes selected on the basis of their expression pattern in normal and tumor samples. A number of candidates were identified as potential diagnostic and therapeutic targets for colorectal tumors and glioblastoma. Several candidate genes were also identified as coding for cell surface cancer/testis antigens. The human cell surfaceome will serve as a reference for further studies aimed at characterizing tumor targets at the surface of human cells. PMID:19805368

  6. Human stem cells for craniomaxillofacial reconstruction.

    PubMed

    Jalali, Morteza; Kirkpatrick, William Niall Alexander; Cameron, Malcolm Gregor; Pauklin, Siim; Vallier, Ludovic

    2014-07-01

    Human stem cell research represents an exceptional opportunity for regenerative medicine and the surgical reconstruction of the craniomaxillofacial complex. The correct architecture and function of the vastly diverse tissues of this important anatomical region are critical for life supportive processes, the delivery of senses, social interaction, and aesthetics. Craniomaxillofacial tissue loss is commonly associated with inflammatory responses of the surrounding tissue, significant scarring, disfigurement, and psychological sequelae as an inevitable consequence. The in vitro production of fully functional cells for skin, muscle, cartilage, bone, and neurovascular tissue formation from human stem cells, may one day provide novel materials for the reconstructive surgeon operating on patients with both hard and soft tissue deficit due to cancer, congenital disease, or trauma. However, the clinical translation of human stem cell technology, including the application of human pluripotent stem cells (hPSCs) in novel regenerative therapies, faces several hurdles that must be solved to permit safe and effective use in patients. The basic biology of hPSCs remains to be fully elucidated and concerns of tumorigenicity need to be addressed, prior to the development of cell transplantation treatments. Furthermore, functional comparison of in vitro generated tissue to their in vivo counterparts will be necessary for confirmation of maturity and suitability for application in reconstructive surgery. Here, we provide an overview of human stem cells in disease modeling, drug screening, and therapeutics, while also discussing the application of regenerative medicine for craniomaxillofacial tissue deficit and surgical reconstruction. PMID:24564584

  7. Paracrine effects of haematopoietic cells on human mesenchymal stem cells

    PubMed Central

    Zhou, Shuanhu

    2015-01-01

    Stem cell function decline during ageing can involve both cell intrinsic and extrinsic mechanisms. Bone and blood formation are intertwined in bone marrow, therefore haematopoietic cells and bone cells could be extrinsic factors for each other. In this study, we assessed the paracrine effects of extrinsic factors from haematopoietic cells on human mesenchymal stem cells (MSCs). Our data showed that haematopoietic cells stimulate proliferation, osteoblast differentiation and inhibit senescence of MSCs; TNF-α, PDGF-β, Wnt1, 4, 6, 7a and 10a, sFRP-3 and sFRP-5 are dominantly expressed in haematopoietic cells; the age-related increase of TNF-α in haematopoietic cells may perform as a negative factor in the interactions of haematopoietic cells on MSCs via TNF-α receptors and then activating NF-κB signaling or Wnt/β-catenin signaling to induce senescence and reduce osteoblast differentiation in MSCs. In conclusion, our data demonstrated that there are paracrine interactions of haematopoietic cells on human MSCs; immunosenescence may be one of the extrinsic mechanisms by which skeletal stem cell function decline during human skeletal ageing. PMID:26030407

  8. Endocrine Cell Clustering During Human Pancreas Development

    PubMed Central

    Jeon, Jongmin; Correa-Medina, Mayrin; Ricordi, Camillo; Edlund, Helena; Diez, Juan A.

    2009-01-01

    The development of efficient, reproducible protocols for directed in vitro differentiation of human embryonic stem (hES) cells into insulin-producing β cells will benefit greatly from increased knowledge regarding the spatiotemporal expression profile of key instructive factors involved in human endocrine cell generation. Human fetal pancreases 7 to 21 weeks of gestational age, were collected following consent immediately after pregnancy termination and processed for immunostaining, in situ hybridization, and real-time RT-PCR expression analyses. Islet-like structures appear from approximately week 12 and, unlike the mixed architecture observed in adult islets, fetal islets are initially formed predominantly by aggregated insulin- or glucagon-expressing cells. The period studied (7–22 weeks) coincides with a decrease in the proliferation and an increase in the differentiation of the progenitor cells, the initiation of NGN3 expression, and the appearance of differentiated endocrine cells. The present study provides a detailed characterization of islet formation and expression profiles of key intrinsic and extrinsic factors during human pancreas development. This information is beneficial for the development of efficient protocols that will allow guided in vitro differentiation of hES cells into insulin-producing cells. (J Histochem Cytochem 57:811–824, 2009) PMID:19365093

  9. Signaling hierarchy regulating human endothelial cell development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

  10. Human embryonic stem cells and lung regeneration

    PubMed Central

    Varanou, A; Page, C P; Minger, S L

    2008-01-01

    Human embryonic stem cells are pluripotent cells derived from the inner cell mass of preimplantation stage embryos. Their unique potential to give rise to all differentiated cell types has generated great interest in stem cell research and the potential that it may have in developmental biology, medicine and pharmacology. The main focus of stem cell research has been on cell therapy for pathological conditions with no current methods of treatment, such as neurodegenerative diseases, cardiac pathology, retinal dysfunction and lung and liver disease. The overall aim is to develop methods of application either of pure cell populations or of whole tissue parts to the diseased organ under investigation. In the field of pulmonary research, studies using human embryonic stem cells have succeeded in generating enriched cultures of type II pneumocytes in vitro. On account of their potential of indefinite proliferation in vitro, embryonic stem cells could be a source of an unlimited supply of cells available for transplantation and for use in gene therapy. Uncovering the ability to generate such cell types will expand our understanding of biological processes to such a degree that disease understanding and management could change dramatically. PMID:18724383

  11. Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

    PubMed

    Itani, Hana A; McMaster, William G; Saleh, Mohamed A; Nazarewicz, Rafal R; Mikolajczyk, Tomasz P; Kaszuba, Anna M; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E; Chen, Wei; Bonami, Rachel H; Marshall, Andrew F; Poffenberger, Greg; Weyand, Cornelia M; Madhur, Meena S; Moore, Daniel J; Harrison, David G; Guzik, Tomasz J

    2016-07-01

    Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II. PMID:27217403

  12. Intrinsic radiation resistance in human chondrosarcoma cells

    SciTech Connect

    Moussavi-Harami, Farid; Mollano, Anthony; Martin, James A.; Ayoob, Andrew; Domann, Frederick E.; Gitelis, Steven; Buckwalter, Joseph A. . E-mail: joseph-buckwalter@uiowa.edu

    2006-07-28

    Human chondrosarcomas rarely respond to radiation treatment, limiting the options for eradication of these tumors. The basis of radiation resistance in chondrosarcomas remains obscure. In normal cells radiation induces DNA damage that leads to growth arrest or death. However, cells that lack cell cycle control mechanisms needed for these responses show intrinsic radiation resistance. In previous work, we identified immortalized human chondrosarcoma cell lines that lacked p16{sup ink4a}, one of the major tumor suppressor proteins that regulate the cell cycle. We hypothesized that the absence of p16{sup ink4a} contributes to the intrinsic radiation resistance of chondrosarcomas and that restoring p16{sup ink4a} expression would increase their radiation sensitivity. To test this we determined the effects of ectopic p16{sup ink4a} expression on chondrosarcoma cell resistance to low-dose {gamma}-irradiation (1-5 Gy). p16{sup ink4a} expression significantly increased radiation sensitivity in clonogenic assays. Apoptosis did not increase significantly with radiation and was unaffected by p16{sup ink4a} transduction of chondrosarcoma cells, indicating that mitotic catastrophe, rather than programmed cell death, was the predominant radiation effect. These results support the hypothesis that p16{sup ink4a} plays a role in the radiation resistance of chondrosarcoma cell lines and suggests that restoring p16 expression will improve the radiation sensitivity of human chondrosarcomas.

  13. Engineering tissue from human embryonic stem cells

    PubMed Central

    Metallo, CM; Azarin, SM; Ji, L; De Pablo, JJ; Palecek, SP

    2008-01-01

    Abstract Recent advances in human embryonic stem cell (hESC) biology now offer an alternative cell source for tissue engineers, as these cells are capable of proliferating indefinitely and differentiating to many clinically relevant cell types. Novel culture methods capable of exerting spatial and temporal control over the stem cell microenvironment allow for more efficient expansion of hESCs, and significant advances have been made toward improving our understanding of the biophysical and biochemical cues that direct stem cell fate choices. Effective production of lineage specific progenitors or terminally differentiated cells enables researchers to incorporate hESC derivatives into engineered tissue constructs. Here, we describe current efforts using hESCs as a cell source for tissue engineering applications, highlighting potential advantages of hESCs over current practices as well as challenges which must be overcome. PMID:18194458

  14. Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice

    PubMed Central

    Wege, Anja K; Schmidt, Marcus; Ueberham, Elke; Ponnath, Marvin; Ortmann, Olaf; Brockhoff, Gero; Lehmann, Jörg

    2014-01-01

    Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγnull (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4+ T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy. PMID:24870377

  15. Clinical translation of human neural stem cells

    PubMed Central

    2013-01-01

    Human neural stem cell transplants have potential as therapeutic candidates to treat a vast number of disorders of the central nervous system (CNS). StemCells, Inc. has purified human neural stem cells and developed culture conditions for expansion and banking that preserve their unique biological properties. The biological activity of these human central nervous system stem cells (HuCNS-SC®) has been analyzed extensively in vitro and in vivo. When formulated for transplantation, the expanded and cryopreserved banked cells maintain their stem cell phenotype, self-renew and generate mature oligodendrocytes, neurons and astrocytes, cells normally found in the CNS. In this overview, the rationale and supporting data for pursuing neuroprotective strategies and clinical translation in the three components of the CNS (brain, spinal cord and eye) are described. A phase I trial for a rare myelin disorder and phase I/II trial for spinal cord injury are providing intriguing data relevant to the biological properties of neural stem cells, and the early clinical outcomes compel further development. PMID:23987648

  16. Clinical translation of human neural stem cells.

    PubMed

    Tsukamoto, Ann; Uchida, Nobuko; Capela, Alexandra; Gorba, Thorsten; Huhn, Stephen

    2013-01-01

    Human neural stem cell transplants have potential as therapeutic candidates to treat a vast number of disorders of the central nervous system (CNS). StemCells, Inc. has purified human neural stem cells and developed culture conditions for expansion and banking that preserve their unique biological properties. The biological activity of these human central nervous system stem cells (HuCNS-SC®) has been analyzed extensively in vitro and in vivo. When formulated for transplantation, the expanded and cryopreserved banked cells maintain their stem cell phenotype, self-renew and generate mature oligodendrocytes, neurons and astrocytes, cells normally found in the CNS. In this overview, the rationale and supporting data for pursuing neuroprotective strategies and clinical translation in the three components of the CNS (brain, spinal cord and eye) are described. A phase I trial for a rare myelin disorder and phase I/II trial for spinal cord injury are providing intriguing data relevant to the biological properties of neural stem cells, and the early clinical outcomes compel further development. PMID:23987648

  17. Rotating cell culture systems for human cell culture: human trophoblast cells as a model.

    PubMed

    Zwezdaryk, Kevin J; Warner, Jessica A; Machado, Heather L; Morris, Cindy A; Höner zu Bentrup, Kerstin

    2012-01-01

    The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines support our understanding of invasion of the uterine wall and remodeling of uterine spiral arteries by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS. The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies. The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS

  18. Cell mechanics and human disease states

    NASA Astrophysics Data System (ADS)

    Suresh, Subra

    2006-03-01

    This presentation will provide summary of our very recent studies exploring the effects of biochemical factors, influenced by foreign organisms or in vivo processes, on intracellular structural reorganization, single-cell mechanical response and motility of a population of cells in the context of two human diseases: malaria induced by Plasmodium falciparum merozoites that invade red blood cells, and gastrointestinal cancer metastasis involving epithelial cells. In both cases, particular attention will be devoted to systematic changes induced in specific molecular species in response to controlled alterations in disease state. The role of critical proteins in influencing the mechanical response of human red bloods during the intra-erythrocytic development of P. falciparum merozoites has also been assessed quantitatively using specific protein knock-out experiments by recourse to gene inactivation methods. Single-cell mechanical response characterization entails such tools as optical tweezers and mechanical plate stretchers whereas cell motility assays and cell-population biorheology characterization involves microfluidic channels. The experimental studies are accompanied by three-dimensional computational simulations at the continuum and mesoscopic scales of cell deformation. An outcome of such combined experimental and computational biophysical studies is the realization of how chemical factors influence single-cell mechanical response, cytoadherence, the biorheology of a large population of cells through microchannels representative of in vivo conditions, and the onset and progression of disease states.

  19. Gammaherpesvirus Infection of Human Neuronal Cells

    PubMed Central

    Jha, Hem Chandra; Mehta, Devan; Lu, Jie; El-Naccache, Darine; Shukla, Sanket K.; Kovacsics, Colleen; Kolson, Dennis

    2015-01-01

    ABSTRACT Gammaherpesviruses human herpesvirus 4 (HHV4) and HHV8 are two prominent members of the herpesvirus family associated with a number of human cancers. HHV4, also known as Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus prevalent in 90 to 95% of the human population, is clinically associated with various neurological diseases such as primary central nervous system lymphoma, multiple sclerosis, Alzheimer’s disease, cerebellar ataxia, and encephalitis. However, the possibility that EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV) can directly infect neurons has been largely overlooked. This study has, for the first time, characterized EBV infection in neural cell backgrounds by using the Sh-Sy5y neuroblastoma cell line, teratocarcinoma Ntera2 neurons, and primary human fetal neurons. Furthermore, we also demonstrated KSHV infection of neural Sh-Sy5y cells. These neuronal cells were infected with green fluorescent protein-expressing recombinant EBV or KSHV. Microscopy, genetic analysis, immunofluorescence, and Western blot analyses for specific viral antigens supported and validated the infection of these cells by EBV and KSHV and showed that the infection was efficient and productive. Progeny virus produced from infected neuronal cells efficiently infected fresh neuronal cells, as well as peripheral blood mononuclear cells. Furthermore, acyclovir was effective at inhibiting the production of virus from neuronal cells similar to lymphoblastoid cell lines; this suggests active lytic replication in infected neurons in vitro. These studies represent a potentially new in vitro model of EBV- and KSHV-associated neuronal disease development and pathogenesis. PMID:26628726

  20. Capsaicin induces immunogenic cell death in human osteosarcoma cells

    PubMed Central

    Jin, Tao; Wu, Hongyan; Wang, Yanlin; Peng, Hao

    2016-01-01

    Immunogenic cell death (ICD) is characterized by the early surface exposure of calreticulin (CRT). As a specific signaling molecule, CRT on the surface of apoptotic tumor cells mediates the recognition and phagocytosis of tumor cells by antigen presenting cells. To date, only a small quantity of anti-cancer chemicals have been found to induce ICD, therefore it is clinically important to identify novel chemicals that may induce ICD. The purpose of the present study is to explore the function of capsaicin in inducing ICD. In the current study, MTT assays were used to examine the growth inhibiting effects of MG-63 cells when they were treated with capsaicin or cisplatin. Mitochondrial membrane potential and western blot analysis were used to investigate capsaicin- and cisplatin-induced apoptosis. In addition, the effects of capsaicin and cisplatin were evaluated for their abilities in inducing calreticulin membrane translocation and mediating ICD in human osteosarcoma cells (MG-63). The results demonstrated that capsaicin and cisplatin can induce the apoptosis of MG-63 cells. However, only capsaicin induced a rapid translocation of CRT from the intracellular space to the cell surface. Treatment with capsaicin increased phagocytosis of MG-63 cells by dendritic cells (DCs), and these MG-63-loaded DCs could efficiently stimulate the secretion of IFN-γ by lymphocytes. These results identify capsaicin as an anti-cancer agent capable of inducing ICD in human osteosarcoma cells in vitro. PMID:27446273

  1. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various...

  2. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various...

  3. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various...

  4. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various...

  5. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various...

  6. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    PubMed Central

    Fukusumi, Hayato; Shofuda, Tomoko; Bamba, Yohei; Yamamoto, Atsuyo; Kanematsu, Daisuke; Handa, Yukako; Okita, Keisuke; Nakamura, Masaya; Yamanaka, Shinya; Okano, Hideyuki; Kanemura, Yonehiro

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes. PMID:27212953

  7. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins.

    PubMed

    Fukusumi, Hayato; Shofuda, Tomoko; Bamba, Yohei; Yamamoto, Atsuyo; Kanematsu, Daisuke; Handa, Yukako; Okita, Keisuke; Nakamura, Masaya; Yamanaka, Shinya; Okano, Hideyuki; Kanemura, Yonehiro

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes. PMID:27212953

  8. Induced pluripotency of human prostatic epithelial cells.

    PubMed

    Zhao, Hongjuan; Sun, Ning; Young, Sarah R; Nolley, Rosalie; Santos, Jennifer; Wu, Joseph C; Peehl, Donna M

    2013-01-01

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that

  9. Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing.

    PubMed

    Tsioris, Konstantinos; Gupta, Namita T; Ogunniyi, Adebola O; Zimnisky, Ross M; Qian, Feng; Yao, Yi; Wang, Xiaomei; Stern, Joel N H; Chari, Raj; Briggs, Adrian W; Clouser, Christopher R; Vigneault, Francois; Church, George M; Garcia, Melissa N; Murray, Kristy O; Montgomery, Ruth R; Kleinstein, Steven H; Love, J Christopher

    2015-12-01

    West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases. PMID:26481611

  10. Cell-in-cell structures are involved in the competition between cells in human tumors.

    PubMed

    Sun, Qiang; Huang, Hongyan; Overholtzer, Michael

    2015-01-01

    The engulfment of live cells may represent a mechanism of cell death. We reported that E-cadherin (epithelial cadherin) expression in human cancer cells favors the formation of cell-in-cell structures through the mechanism known as entosis, and that entosis contributes to a form of cellular competition in heterogeneous cancer cell populations. PMID:27308493

  11. Cell-in-cell structures are involved in the competition between cells in human tumors

    PubMed Central

    Sun, Qiang; Huang, Hongyan; Overholtzer, Michael

    2015-01-01

    The engulfment of live cells may represent a mechanism of cell death. We reported that E-cadherin (epithelial cadherin) expression in human cancer cells favors the formation of cell-in-cell structures through the mechanism known as entosis, and that entosis contributes to a form of cellular competition in heterogeneous cancer cell populations. PMID:27308493

  12. CLOSTRIDIUM SPORE ATTACHMENT TO HUMAN CELLS

    SciTech Connect

    PANESSA-WARREN,B.; TORTORA,G.; WARREN,J.

    1997-08-10

    This paper uses high resolution scanning electron microscopy (SEM) with a LaB6 gun and the newest commercial field emission guns, to obtain high magnification images of intact clostridial spores throughout the activation/germination/outgrowth process. By high resolution SEM, the clostridial exosporial membrane can be seen to produce numerous delicate projections (following activation), that extend from the exosporial surface to a nutritive substrate (agar), or cell surface when anaerobically incubated in the presence of human cells (embryonic fibroblasts and colon carcinoma cells). Magnifications of 20,000 to 200,000Xs at accelerating voltages low enough to minimize or eliminate specimen damage (1--5 kV) have permitted the entire surface of C.sporogenes and C.difficile endospores to be examined during all stages of germination. The relationships between the spore and the agar or human cell surface were also clearly visible.

  13. Phospholipid composition of cultured human endothelial cells.

    PubMed

    Murphy, E J; Joseph, L; Stephens, R; Horrocks, L A

    1992-02-01

    Detailed analyses of the phospholipid compositions of cultured human endothelial cells are reported here. No significant differences were found between the phospholipid compositions of cells from human artery, saphenous and umbilical vein. However, due to the small sample sizes, relatively large standard deviations for some of the phospholipid classes were observed. A representative composition of endothelial cells is: phosphatidylcholine 36.6%, choline plasmalogen 3.7%, phosphatidylethanolamine 10.2%, ethanolamine plasmalogen 7.6%, sphingomyelin 10.8%, phosphatidylserine 7.1%, lysophosphatidylcholine 7.5%, phosphatidylinositol 3.1%, lysophosphatidylethanolamine 3.6%, phosphatidylinositol 4,5-bisphosphate 1.8%, phosphatidic acid 1.9%, phosphatidylinositol 4-phosphate 1.5%, and cardiolipin 1.9%. The cells possess high choline plasmalogen and lysophosphatidylethanolamine contents. The other phospholipids are within the normal biological ranges expected. Phospholipids were separated by high-performance liquid chromatography and quantified by lipid phosphorus assay. PMID:1315902

  14. A human gallbladder adenocarcinoma cell line.

    PubMed

    Morgan, R T; Woods, L K; Moore, G E; McGavran, L; Quinn, L A; Semple, T U

    1981-06-01

    A continuous cell line, COLO 346, was established from a liver metastasis in a patient with adenocarcinoma of the gallbladder. COLO 346 grew as an adherent monolayer of pleomorphic epithelioid cells. COLO 346 cells produced esterone, but no estradiol, progesterone, or cortisol. No adrenocorticotropic hormones, beta-subunit of human chorionic gonadotropin, carcinoembryonic antigen, or alpha-fetoprotein production by the cells was detected. Cell doubling time was 36 h. Seven allelic isozymes were assayed. COLO 346 had a chromosome mode of 74 at 21 months postestablishment with 6 marker chromosomes present in 100% of the cells analyzed. COLO 346 has been in continuous culture for over 2 yr and is available to other investigators for their studies. PMID:7262900

  15. Human Colon Cancer Cells Cultivated in Space

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Within five days, bioreactor cultivated human colon cancer cells (shown) grown in Microgravity on the STS-70 mission in 1995, had grown 30 times the volume of the control specimens on Earth. The samples grown in space had a higher level of cellular organization and specialization. Because they more closely resemble tumors found in the body, microgravity grown cell cultures are ideal for research purposes.

  16. Neutron irradiation of human melanoma cells.

    PubMed

    Brown, K; Mountford, M H; Allen, B J; Mishima, Y; Ichihashi, M; Parsons, P

    1989-01-01

    The biological characteristics and in vitro radiosensitivity of melanoma cells to thermal neutrons were investigated as a guide to the effectiveness of boron neutron capture therapy. Plateau phase cultures of three human malignant melanoma-established cell lines were examined for cell density at confluence, doubling time, cell cycle parameters, chromosome constitution, and melanin content. Cell survival dose-response curves, for cells preincubated in the presence or absence of p-boronophenylalanine. HCl (10B1-BPA), were measured over the dose range 0.6-8.0 Gy (N + gamma). The neutron fluence rate was 2.6 x 10(9) n/cm2/s and the total dose rate 3.7 Gy/h (31% gamma). Considerable differences were observed in the morphology and cellular properties of the cell lines. Two cell lines (96E and 96L) were amelanotic, and one was melanotic (418). An enhanced killing for neutron irradiation was found only for the melanotic cells after 20 h preincubation with 10 micrograms/ml 10B1-BPA. In view of the doubling times of the cell lines of about 23 h (96E and 96L) or of 36 h (418), it seems likely that an increased boron uptake, and hence increased radiosensitivity, might result if the preincubation period with 10B1-BPA is extended to several hours longer than the respective cell cycle times. PMID:2798324

  17. 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. PMID:25520283

  18. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    SciTech Connect

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  19. 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

  20. 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

  1. B10 cell regulation of health and disease

    PubMed Central

    Candando, Kathleen M.; Lykken, Jacquelyn M.; Tedder, Thomas F.

    2014-01-01

    Summary While B cells are traditionally regarded as promoters of the immune response via antibody secretion and pro-inflammatory cytokine production, recent studies have also confirmed an important role for B-cell-mediated negative regulation of immunity. Tremendous advances in the characterization of the mechanisms by which regulatory B cells function has led to the identification of a novel subset of regulatory B cells known as B10 cells, which regulate immune responses through the production of the anti-inflammatory cytokine interleukin-10 (IL-10). B10 cells are best defined by their functional ability to produce IL-10, as they are not confined to any particular phenotypic subset. B10 cells function in an antigen-specific manner that requires cognate interactions with T cells in vivo to regulate immune responses and have been demonstrated to be potent regulators of allergic and autoimmune disease, cancer, infection, and transplant rejection. Importantly, the recent discovery of human B10 cells has accelerated this field to the forefront of clinical research where the possibility of harnessing the regulatory potential of B10 cells for treatment of aberrant immune responses and diseases may become feasible. PMID:24712471

  2. Large scale cultivation of an anti-D (IgM) antibody producing human/mouse heterohybridoma.

    PubMed

    Fizil, A; Hencsey, Z; Veszely, G; Inzelt-Kovács, M; Bánkúti, L

    1996-01-01

    A human/mouse heterohybridoma cell line secreting anti-Rh(D) antibody was established by the fusion of Epstein-Barr Virus (EBV) transformed human B lymphocytes and human/mouse heteromyelomas. During scaling-up the cell line was adapted to suspension culture, then medium composition was gradually altered into a more economical one. The adapted cell line has kept its multiplication and antibody secreting characteristics in the new medium. Parameters of large scale batch cultivation in bioreactors were optimized in working volume of 3.6 litre and production was carried out under these parameters in working volume of 24 litre. Optimized parameters were the follows: (i) the cell line is highly sensitive to dissolved oxygen (DO) level, its optimal DO is 10%; (ii) optimal inoculum cell count is 3 x 10(5) cell/ml; (iii) Marine-blade impeller was chosen for supporting the requested oxygen transfer. Using the optimized parameters HUMAN Co. Ltd. produces the Monoclonal Anti-D (IgM) reagent, which has been registered in 1995 in Hungary and now it is on the market. PMID:9147726

  3. Advances in Human B Cell Phenotypic Profiling

    PubMed Central

    Kaminski, Denise A.; Wei, Chungwen; Qian, Yu; Rosenberg, Alexander F.; Sanz, Ignacio

    2012-01-01

    To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation (“Big Biology”), necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort. PMID:23087687

  4. Human embryonic stem cells: preclinical perspectives

    PubMed Central

    Deb, Kaushik Dilip; Sarda, Kanchan

    2008-01-01

    Human embryonic stem cells (hESCs) have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic. PMID:18230169

  5. Human norovirus culture in B cells

    PubMed Central

    Jones, Melissa K; Grau, Katrina R; Costantini, Veronica; Kolawole, Abimbola O; de Graaf, Miranda; Freiden, Pamela; Graves, Christina L; Koopmans, Marion; Wallet, Shannon M; Tibbetts, Scott A; Schultz-Cherry, Stacey; Wobus, Christiane E; Vinjé, Jan; Karst, Stephanie M

    2015-01-01

    Human noroviruses (HunoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HunoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HunoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-sydney HunoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HunoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. analysis of infection or attachment samples, including rna extraction and rt-qpcr, requires ~6 h. PMID:26513671

  6. Human norovirus culture in B cells.

    PubMed

    Jones, Melissa K; Grau, Katrina R; Costantini, Veronica; Kolawole, Abimbola O; de Graaf, Miranda; Freiden, Pamela; Graves, Christina L; Koopmans, Marion; Wallet, Shannon M; Tibbetts, Scott A; Schultz-Cherry, Stacey; Wobus, Christiane E; Vinjé, Jan; Karst, Stephanie M

    2015-12-01

    Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h. PMID:26513671

  7. Statins impair glucose uptake in human cells

    PubMed Central

    Nowis, Dominika; Malenda, Agata; Furs, Karolina; Oleszczak, Bozenna; Sadowski, Radoslaw; Chlebowska, Justyna; Firczuk, Malgorzata; Bujnicki, Janusz M; Staruch, Adam D; Zagozdzon, Radoslaw; Glodkowska-Mrowka, Eliza; Szablewski, Leszek; Golab, Jakub

    2014-01-01

    Objective Considering the increasing number of clinical observations indicating hyperglycemic effects of statins, this study was designed to measure the influence of statins on the uptake of glucose analogs by human cells derived from liver, adipose tissue, and skeletal muscle. Design Flow cytometry and scintillation counting were used to measure the uptake of fluorescently labeled or tritiated glucose analogs by differentiated visceral preadipocytes, skeletal muscle cells, skeletal muscle myoblasts, and contact-inhibited human hepatocellular carcinoma cells. A bioinformatics approach was used to predict the structure of human glucose transporter 1 (GLUT1) and to identify the presence of putative cholesterol-binding (cholesterol recognition/interaction amino acid consensus (CRAC)) motifs within this transporter. Mutagenesis of CRAC motifs in SLC2A1 gene and limited proteolysis of membrane GLUT1 were used to determine the molecular effects of statins. Results Statins significantly inhibit the uptake of glucose analogs in all cell types. Similar effects are induced by methyl-β-cyclodextrin, which removes membrane cholesterol. Statin effects can be rescued by addition of mevalonic acid, or supplementation with exogenous cholesterol. Limited proteolysis of GLUT1 and mutagenesis of CRAC motifs revealed that statins induce conformational changes in GLUTs. Conclusions Statins impair glucose uptake by cells involved in regulation of glucose homeostasis by inducing cholesterol-dependent conformational changes in GLUTs. This molecular mechanism might explain hyperglycemic effects of statins observed in clinical trials. PMID:25452863

  8. Androgen receptor in human endothelial cells

    PubMed Central

    Torres-Estay, Verónica; Carreño, Daniela V; San Francisco, Ignacio F; Sotomayor, Paula; Godoy, Alejandro S; Smith, Gary J

    2015-01-01

    Androgen receptor (AR) is a ligand-inducible transcription factor, and a member of the steroid-thyroid-retinoid receptor superfamily, that mediates the biological effects of androgens in a wide range of physiological and pathological processes. AR expression was identified in vascular cells nearly 20 years ago, and recent research has shown that AR mediates a variety of actions of androgens in endothelial and vascular smooth muscle cells. In this mini-review, we review evidence indicating the importance of AR in human endothelial cell (HUVEC) homeostatic and pathogenic processes. Although a role for AR in the modulation of HUVEC biology is evident, the molecular mechanisms by which AR regulates HUVEC homeostasis and disease processes are not fully understood. Understanding these mechanisms could provide critical insights into the processes of pathogenesis of diseases ranging from cardiovascular disease to cancer that are major causes of human morbidity and mortality. PMID:25563353

  9. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz; Rockx, Barry

    2013-03-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  10. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J. Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz

    2013-01-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  11. Diabetes Impairs Stem Cell and Proangiogenic Cell Mobilization in Humans

    PubMed Central

    Fadini, Gian Paolo; Albiero, Mattia; Vigili de Kreutzenberg, Saula; Boscaro, Elisa; Cappellari, Roberta; Marescotti, Mariacristina; Poncina, Nicol; Agostini, Carlo; Avogaro, Angelo

    2013-01-01

    OBJECTIVE Diabetes mellitus (DM) increases cardiovascular risk, at least in part, through shortage of vascular regenerative cells derived from the bone marrow (BM). In experimental models, DM causes morphological and functional BM alterations, but information on BM function in human DM is missing. Herein, we sought to assay mobilization of stem and proangiogenic cells in subjects with and without DM. RESEARCH DESIGN AND METHODS In a prospective trial (NCT01102699), we tested BM responsiveness to 5 μg/kg human recombinant granulocyte colony–stimulating factor (hrG-CSF) in 24 individuals with DM (10 type 1 and 14 type 2) and 14 individuals without DM. Before and 24 h after hrG-CSF, we quantified circulating stem/progenitor cells and total and differential white blood cell counts. We also evaluated in vivo the proangiogenic capacity of peripheral blood mononuclear cells using the Matrigel plug assay. RESULTS In response to hrG-CSF, levels of CD34+ cells and other progenitor cell phenotypes increased in subjects without DM. Patients with DM had significantly impaired mobilization of CD34+, CD133+, and CD34+CD133+ hematopoietic stem cells and CD133+KDR+ endothelial progenitors, independently of potential confounders. The in vivo angiogenic capacity of peripheral blood mononuclear cells significantly increased after hrG-CSF in control subjects without DM, but not in patients with DM. DM was also associated with the inability to upregulate CD26/DPP-4 on CD34+ cells, which is required for the mobilizing effect of granulocyte colony–stimulating factor. CONCLUSIONS Stem and proangiogenic cell mobilization in response to hrG-CSF is impaired in DM, possibly because of maladaptive CD26/DPP-4 regulation. These alterations may hamper tissue repair and favor the development of cardiovascular complications. PMID:23111057

  12. Primary Bioassay of Human Myeloma Stem Cells

    PubMed Central

    Hamburger, Anne; Salmon, Sydney E.

    1977-01-01

    The ability to clone primary tumors in soft agar has proven useful in the study of the kinetics and biological properties of tumor stem cells. We report the development of an in vitro assay which permits formation of colonies of human monoclonal plasma cells in soft agar. Colony growth has been observed from bone marrow aspirates from 75% of the 70 patients with multiple myeloma or related monoclonal disorders studied. Growth was induced with either 0.02 ml of human type O erythrocytes or 0.25 ml of medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. 5-500 colonies appeared after 2-3 wk in culture yielding a plating efficiency of 0.001-0.1%. The number of myeloma colonies was proportional to the number of cells plated between concentrations of 105-106 and back-extrapolated through zero, suggesting that colonies were clones derived from single myeloma stem cells. Morphological, histochemical, and functional criteria showed the colonies to consist of immature plasmablasts and mature plasma cells. 60-80% of cells picked from colonies contained intracytoplasmic monoclonal immunoglobulin. Colony growth was most easily achieved from the bone marrow cells of untreated patients or those in relapse. Only 50% of bone marrow samples from patients in remission were successfully cultured. Tritiated thymidine suicide studies provided evidence that for most myeloma patients, a very high proportion of myeloma colony-forming cells was actively in transit through the cell cycle. Velocity sedimentation at 1 g showed myeloma stem cells sedimented in a broad band with a peak at 13 mm/h. Antibody to granulocyte colony-stimulating factor did not reduce the number or size of the colonies. Increased numbers of myeloma colonies were seen when the marrow was depleted of colony-stimulating factor elaborating adherent cells before plating. This bioassay should prove useful in studying the in vitro biological behavior of certain bone marrow-derived (B)-cell

  13. How to make a human germ cell.

    PubMed

    Cooke, Paul S; Nanjappa, Manjunatha K

    2015-01-01

    How the primordial germ cell (PGC) lineage, which eventually gives rise to spermatozoa in males and oocytes in females, is established in the developing mammalian embryo has been a critical topic in both developmental and reproductive biology for many years. There have been significant breakthroughs over the past two decades in establishing both the source of PGCs and the factors that regulate the specification of this lineage in mice, [1] but our understanding of the factors that control PGC development in the human is rudimentary. The SRY-related HMG-box (SOX) family of transcription factors consists of 20 genes in humans and mice that are involved in the maintenance of pluripotency, male sexual development, and other processes. A recent paper in Cell has identified one member of this family, SOX17, as an essential factor for inducing the PGC lineage in humans. [2] Surprisingly, this protein does not appear to have a role in PGC specification in mice. This work not only introduces a new and important player to the field of germ cell specification, but also emphasizes the uniqueness of human PGC development compared to more extensively studied mouse models. PMID:25791734

  14. DNA repair responses in human skin cells

    SciTech Connect

    Hanawalt, P.C.; Liu, S.C.; Parsons, C.S.

    1981-07-01

    Sunlight and some environmental chemical agents produce lesions in the DNA of human skin cells that if unrepaired may interfere with normal functioning of these cells. The most serious outcome of such interactions may be malignancy. It is therefore important to develop an understanding of mechanisms by which the lesions may be repaired or tolerated without deleterious consequences. Our models for the molecular processing of damaged DNA have been derived largely from the study of bacterial systems. Some similarities but significant differences are revealed when human cell responses are tested against these models. It is also of importance to learn DNA repair responses of epidermal keratinocytes for comparison with the more extensive studies that have been carried out with dermal fibroblasts. Our experimental results thus far indicate similarities for the excision-repair of ultraviolet-induced pyrimidine dimers in human keratinocytes and fibroblasts. Both the monoadducts and the interstrand crosslinks produced in DNA by photoactivated 8-methoxypsoralen (PUVA) can be repaired in normal human fibroblasts but not in those from xeroderma pigmentosum patients. The monoadducts, like pyrimidine dimers, are probably the more mutagenic/carcinogenic lesions while the crosslinks are less easily repaired and probably result in more effective blocking of DNA function. It is suggested that a split-dose protocol that maximizes the production of crosslinks while minimizing the yield of monoadducts may be more effective and potentially less carcinogenic than the single ultraviolet exposure regimen in PUVA therapy for psoriasis.

  15. Genome Editing in Human Pluripotent Stem Cells.

    PubMed

    Smith, Cory; Ye, Zhaohui; Cheng, Linzhao

    2016-01-01

    Pluripotent stem cells (PSCs), defined by their capacity for self-renewal and differentiation into all cell types, are an integral tool for basic biological research and disease modeling. However, full use of PSCs for research and regenerative medicine requires the ability to precisely edit their DNA to correct disease-causing mutations and for functional analysis of genetic variations. Recent advances in DNA editing of human stem cells (including PSCs) have benefited from the use of designer nucleases capable of making double-strand breaks (DSBs) at specific sequences that stimulate endogenous DNA repair. The clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become the preferred designer nuclease for genome editing in human PSCs and other cell types. Here we describe the principles for designing a single guide RNA to uniquely target a gene of interest and describe strategies for disrupting, inserting, or replacing a specific DNA sequence in human PSCs. The improvements in efficiency and ease provided by these techniques allow individuals to precisely engineer PSCs in a way previously limited to large institutes and core facilities. PMID:27037079

  16. Gemcitabine induces cell senescence in human pancreatic cancer cell lines.

    PubMed

    Song, Yao; Baba, Tomohisa; Mukaida, Naofumi

    2016-08-26

    Patients with pancreatic ductal adenocarcinoma (PDAC) commonly require chemotherapy because they frequently develop metastatic disease or locally advanced tumors. Gemcitabine, an analogue of cytosine arabinoside, is commonly used for PDAC treatment. We observed that gemcitabine induced senescence phenotypes characterized by enhanced senescence-associated β-galactosidase (SA β-Gal) staining and increased expression of senescence-associated molecules in two human pancreatic cancer cell lines, Miapaca-2 and Panc-1, which exhibit resistance to gemcitabine but not L3.pl cells with a high sensitivity to gemcitabine. Gemcitabine-induced cell senescence can be inhibited by reactive oxygen species inhibitor, N-acetyl cysteine. Although gemcitabine also enhanced CXCL8 expression, anti-CXCL8 antibody failed to reduce gemcitabine-induced increases in SA β-Gal-positive cell numbers. These observations would indicate that cell senescence can proceed independently of CXCL8 expression, a characteristic feature of senescence-associated secretion phenotype. PMID:27311854

  17. Cloning of human lung cancer cells.

    PubMed Central

    Walls, G. A.; Twentyman, P. R.

    1985-01-01

    We have carried out a comparison of two different methods for cloning human lung cancer cells. The method of Courtenay & Mills (1978) generally gave higher plating efficiencies (PE) than the method of Carney et al. (1980). The number of colonies increased with incubation time in both methods and the weekly medium replenishment in the Courtenay method was advantageous for longer incubation times of several weeks. In the Courtenay method, the use of August rat red blood cells (RBC) and low oxygen tension were both found to be necessary factors for maximum plating efficiency. The usefulness of heavily irradiated feeder cells in improving PE is less certain; each cell type may have its own requirement. PMID:3904799

  18. Differentiation of human innate lymphoid cells (ILCs).

    PubMed

    Juelke, Kerstin; Romagnani, Chiara

    2016-02-01

    During the last years, a high complexity in innate lymphoid lineages now collectively referred to as innate lymphoid cells (ILCs) has been revealed. ILCs can be grouped according to their effector functions and transcriptional requirements into three main groups, termed group 1, 2 and 3 ILCs. The differentiation of ILC lineages from hematopoietic precursors and the molecular switches guiding their developmental fate have started to be characterized both in mice and humans. In this review, we discuss the origin, differentiation stages and plasticity of human ILC subsets as well as the signals that drive ILC lineage commitment and acquisition of their unique effector programs. PMID:26707651

  19. High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry

    PubMed Central

    Nair, Nitya; Newell, Evan W.; Vollmers, Christopher; Quake, Stephen R.; Morton, John M.; Davis, Mark M.; He, Xiao-Song; Greenberg, Harry B.

    2015-01-01

    In-depth phenotyping of human intestinal antibody secreting cells (ASCs) and their precursors is important for developing improved mucosal vaccines. We used single-cell mass cytometry to simultaneously analyze 34 differentiation and trafficking markers on intestinal and circulating B cells. In addition, we labeled rotavirus double-layered particles with a metal isotope and characterized B cells specific to the rotavirus VP6 major structural protein. We describe the heterogeneity of the intestinal B cell compartment, dominated by ASCs with some phenotypic and transcriptional characteristics of long-lived plasma cells. Using principal component analysis, we visualized the phenotypic relationships between major B cell subsets in the intestine and blood, and revealed that IgM+ memory B cells (MBCs) and naïve B cells were phenotypically related as were CD27− MBCs and switched MBCs. ASCs in the intestine and blood were highly clonally related, but associated with distinct trajectories of phenotypic development. VP6-specific B cells were present among diverse B cell subsets in immune donors, including naïve B cells, with phenotypes representative of the overall B cell pool. These data provide a high dimensional view of intestinal B cells and the determinants regulating humoral memory to a ubiquitous, mucosal pathogen at steady-state. PMID:25899688

  20. High prevalence of side population in human cancer cell lines

    PubMed Central

    Boesch, Maximilian; Zeimet, Alain G.; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems. PMID:27226981

  1. Lymphoid Cell-Glioma Cell Interaction Enhances Cell Coat Production by Human Gliomas: Novel Suppressor Mechanism

    NASA Astrophysics Data System (ADS)

    Dick, Steven J.; Macchi, Beatrice; Papazoglou, Savvas; Oldfield, Edward H.; Kornblith, Paul L.; Smith, Barry H.; Gately, Maurice K.

    1983-05-01

    Certain human glioma lines produce mucopolysaccharide coats that impair the generation of cytolytic lymphocytes in response to these lines in vitro. Coat production is substantially enhanced by the interaction of glioma cells with a macromolecular factor released by human peripheral blood mononuclear cells in culture. This interaction thus constitutes an unusual mechanism by which inflammatory cells may nonspecifically suppress the cellular immune response to at least one class of solid tumors in humans.

  2. Human Olfactory Mucosa Multipotent Mesenchymal Stromal Cells Promote Survival, Proliferation, and Differentiation of Human Hematopoietic Cells

    PubMed Central

    Diaz-Solano, Dylana; Wittig, Olga; Ayala-Grosso, Carlos; Pieruzzini, Rosalinda

    2012-01-01

    Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit–granulocyte/macrophage (CFU-GM) progenitors and CD34+ cells were found, at 43 days of co-culture. Reverse transcriptase–polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines. PMID:22471939

  3. The first recombinant human coagulation factor VIII of human origin: human cell line and manufacturing characteristics

    PubMed Central

    Casademunt, Elisabeth; Martinelle, Kristina; Jernberg, Mats; Winge, Stefan; Tiemeyer, Maya; Biesert, Lothar; Knaub, Sigurd; Walter, Olaf; Schröder, Carola

    2012-01-01

    Introduction Since the early 1990s, recombinant human clotting factor VIII (rhFVIII) produced in hamster cells has been available for haemophilia A treatment. However, the post-translational modifications of these proteins are not identical to those of native human FVIII, which may lead to immunogenic reactions and the development of inhibitors against rhFVIII. For the first time, rhFVIII produced in a human host cell line is available. Aim We describe here the establishment of the first human production cell line for rhFVIII and the manufacturing process of this novel product. Methods and results A human cell line expressing rhFVIII was derived from human embryonic kidney (HEK) 293 F cells transfected with an FVIII expression plasmid. No virus or virus-like particles could be detected following extensive testing. The stringently controlled production process is completely free from added materials of animal or human origin. Multistep purification employing a combination of filtration and chromatography steps ensures the efficient removal of impurities. Solvent/detergent treatment and a 20 nm pore size nanofiltration step, used for the first time in rhFVIII manufacturing, efficiently eliminate any hypothetically present viruses. In contrast to hamster cell-derived products, this rhFVIII product does not contain hamster-like epitopes, which might be expected to be immunogenic. Conclusions HEK 293 F cells, whose parental cell line HEK 293 has been used by researchers for decades, are a suitable production cell line for rhFVIII and will help avoid immunogenic epitopes. A modern manufacturing process has been developed to ensure the highest level of purity and pathogen safety. PMID:22690791

  4. Characterization of Human Astrovirus Cell Entry

    PubMed Central

    Méndez, Ernesto; Muñoz-Yañez, Claudia; Sánchez-San Martín, Claudia; Aguirre-Crespo, Gabriela; Baños-Lara, M. del Rocio; Gutierrez, Michelle; Espinosa, Rafaela; Acevedo, Yunuén; Arias, Carlos F.

    2014-01-01

    Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle. PMID:24335315

  5. Inner Ear Hair Cell-Like Cells from Human Embryonic Stem Cells

    PubMed Central

    Ronaghi, Mohammad; Nasr, Marjan; Ealy, Megan; Durruthy-Durruthy, Robert; Waldhaus, Joerg; Diaz, Giovanni H.; Joubert, Lydia-Marie; Oshima, Kazuo

    2014-01-01

    In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation. PMID:24512547

  6. Inner ear hair cell-like cells from human embryonic stem cells.

    PubMed

    Ronaghi, Mohammad; Nasr, Marjan; Ealy, Megan; Durruthy-Durruthy, Robert; Waldhaus, Joerg; Diaz, Giovanni H; Joubert, Lydia-Marie; Oshima, Kazuo; Heller, Stefan

    2014-06-01

    In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation. PMID:24512547

  7. Cell Culture Assay for Human Noroviruses [response

    SciTech Connect

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  8. Immortalization of human myogenic progenitor cell clone retaining multipotentiality

    SciTech Connect

    Hashimoto, Naohiro . E-mail: nao@nils.go.jp; Kiyono, Tohru; Wada, Michiko R.; Shimizu, Shirabe; Yasumoto, Shigeru; Inagawa, Masayo

    2006-10-06

    Human myogenic cells have limited ability to proliferate in culture. Although forced expression of telomerase can immortalize some cell types, telomerase alone delays senescence of human primary cultured myogenic cells, but fails to immortalize them. In contrast, constitutive expression of both telomerase and the E7 gene from human papillomavirus type 16 immortalizes primary human myogenic cells. We have established an immortalized primary human myogenic cell line preserving multipotentiality by ectopic expression of telomerase and E7. The immortalized human myogenic cells exhibit the phenotypic characteristics of their primary parent, including an ability to undergo myogenic, osteogenic, and adipogenic terminal differentiation under appropriate culture conditions. The immortalized cells will be useful for both basic and applied studies aimed at human muscle disorders. Furthermore, immortalization by transduction of telomerase and E7 represents a useful method by which to expand human myogenic cells in vitro without compromising their ability to differentiate.

  9. Proteoglycans from human umbilical vein endothelial cells.

    PubMed

    Griesmacher, A; Hennes, R; Keller, R; Greiling, H

    1987-10-01

    Human umbilical vein endothelial cells were incubated with [35S]sulphate and investigated for their proteoglycan production. By gel chromatography, ion-exchange chromatography and CsCl density-gradient centrifugation we obtained preparative amounts of the endothelial proteoheparan sulphate HSI and of proteochondroitin sulphate from the conditioned medium of mass-cultured human umbilical vein endothelial cells. Approximately 90% of the 35S-labeled material in the endothelial cell conditioned medium was proteochondroitin sulphate. This molecule, with a molecular mass of 180-200 kDa, contains four side-chains of 35-40 kDa and a core protein of 35-40 kDa. Two proteoheparan sulphate forms (HSI and HSII) from the conditioned medium were distinguished by molecular mass and transport kinetics from the cell layer to the medium in pulse-chase experiments. One major form (HSI), with an approximate molecular mass of 160-200 kDa a core protein of 55-60 kDa and three to four polysaccharide side-chains of 35 kDa each, was found enriched in the cellular membrane pellet. Another proteoheparan sulphate (HSII), with polysaccharide moieties of 20 kDa, is enriched in the subendothelial matrix (substratum). PMID:2959475

  10. Umbilical cord mesenchymal stem cells: adjuvants for human cell transplantation.

    PubMed

    Friedman, Robb; Betancur, Monica; Boissel, Laurent; Tuncer, Hande; Cetrulo, Curtis; Klingemann, Hans

    2007-12-01

    The Wharton's jelly of the umbilical cord is rich in mesenchymal stem cells (UC-MSCs) that fulfill the criteria for MSCs. Here we describe a novel, simple method of obtaining and cryopreserving UC-MSCs by extracting the Wharton's jelly from a small piece of cord, followed by mincing the tissue and cryopreserving it in autologous cord plasma to prevent exposure to allogeneic or animal serum. This direct freezing of cord microparticles without previous culture expansion allows the processing and freezing of umbilical cord blood (UCB) and UC-MSCs from the same individual on the same day on arrival in the laboratory. UC-MSCs produce significant concentrations of hematopoietic growth factors in culture and augment hematopoietic colony formation when co-cultured with UCB mononuclear cells. Mice undergoing transplantation with limited numbers of human UCB cells or CD34(+) selected cells demonstrated augmented engraftment when UC-MSCs were co-transplanted. We also explored whether UC-MSCs could be further manipulated by transfection with plasmid-based vectors. Electroporation was used to introduce cDNA and mRNA constructs for GFP into the UC-MSCs. Transfection efficiency was 31% for cDNA and 90% for mRNA. These data show that UC-MSCs represent a reliable, easily accessible, noncontroversial source of MSCs. They can be prepared and cryopreserved under good manufacturing practices (GMP) conditions and are able to enhance human hematopoietic engraftment in SCID mice. Considering their cytokine production and their ability to be easily transfected with plasmid-based vectors, these cells should have broad applicability in human cell-based therapies. PMID:18022578

  11. Human Embryonic Stem Cells and Cardiac Repair

    PubMed Central

    Zhu, Wei-Zhong; Hauch, Kip; Xu, Chunhui; Laflamme, Michael A.

    2008-01-01

    The muscle lost after a myocardial infarction is replaced with non-contractile scar tissue, often initiating heart failure. Whole-organ cardiac transplantation is the only currently available clinical means of replacing the lost muscle, but this option is limited by the inadequate supply of donor hearts. Thus, cell-based cardiac repair has attracted considerable interest as an alternative means of ameliorating cardiac injury. Because of their tremendous capacity for expansion and unquestioned cardiac potential, pluripotent human embryonic stem cells (hESCs) represent an attractive candidate cell source for obtaining cardiomyocytes and other useful mesenchymal cell types for such therapies. hESC-derived cardiomyocytes (hESC-CMs) exhibit a committed cardiac phenotype and robust proliferative capacity, and recent testing in rodent infarct models indicates that they can partially remuscularize injured hearts and improve contractile function. Although the latter successes give good reason for optimism, considerable challenges remain to the successful application of hESCs to cardiac repair, including the need for preparations of high cardiac purity, improved methods of delivery, and approaches to overcome immune rejection and other causes of graft cell death. This review will describe the phenotype of hESC-CMs and preclinical experience with these cells and will consider strategies to overcoming the aforementioned challenges. PMID:18657407

  12. Human cell culture in a space bioreactor

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.

    1988-01-01

    Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

  13. Human proximal tubule cells form functional microtissues.

    PubMed

    Prange, Jenny A; Bieri, Manuela; Segerer, Stephan; Burger, Charlotte; Kaech, Andres; Moritz, Wolfgang; Devuyst, Olivier

    2016-04-01

    The epithelial cells lining the proximal tubules of the kidney mediate complex transport processes and are particularly vulnerable to drug toxicity. Drug toxicity studies are classically based on two-dimensional cultures of immortalized proximal tubular cells. Such immortalized cells are dedifferentiated, and lose transport properties (including saturable endocytic uptake) encountered in vivo. Generating differentiated, organotypic human microtissues would potentially alleviate these limitations and facilitate drug toxicity studies. Here, we describe the generation and characterization of kidney microtissues from immortalized (HK-2) and primary (HRPTEpiC) human renal proximal tubular epithelial cells under well-defined conditions. Microtissue cultures were done in hanging drop GravityPLUS™ culture plates and were characterized for morphology, proliferation and differentiation markers, and by monitoring the endocytic uptake of albumin. Kidney microtissues were successfully obtained by co-culturing HK-2 or HRPTEpiC cells with fibroblasts. The HK-2 microtissues formed highly proliferative, but dedifferentiated microtissues within 10 days of culture, while co-culture with fibroblasts yielded spherical structures already after 2 days. Low passage HRPTEpiC microtissues (mono- and co-culture) were less proliferative and expressed tissue-specific differentiation markers. Electron microscopy evidenced epithelial differentiation markers including microvilli, tight junctions, endosomes, and lysosomes in the co-cultured HRPTEpiC microtissues. The co-cultured HRPTEpiC microtissues showed specific uptake of albumin that could be inhibited by cadmium and gentamycin. In conclusion, we established a reliable hanging drop protocol to obtain functional kidney microtissues with proximal tubular epithelial cell lines. These microtissues could be used for high-throughput drug and toxicology screenings, with endocytosis as a functional readout. PMID:26676951

  14. Human somatic cell nuclear transfer is alive and well.

    PubMed

    Cibelli, Jose B

    2014-06-01

    In this issue, Chung et al. (2014) generate human embryonic stem cells by fusing an adult somatic cell to a previously enucleated human oocyte, in agreement with recent reports by the Mitalipov and Egli groups. We can now safely say that human somatic cell nuclear transfer is alive and well. PMID:24905159

  15. 3 CFR - Guidelines for Human Stem Cell Research

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Guidelines apply to the expenditure of NIH funds for research using human embryonic stem cells and certain uses of human induced pluripotent stem cells. The Guidelines are based on the principles that responsible research with human embryonic stem cells has the potential to improve our understanding of...

  16. Human umbilical cord perivascular cells (HUCPVC)

    PubMed Central

    Zebardast, Nazlee; Lickorish, David

    2010-01-01

    Human bone marrow mesenchymal stem cells (hBM-MSC) have recently been employed in the clinical treatment of challenging skin defects. We have described an MSC population that can be easily harvested from human umbilical cord perivascular tissue, human umbilical cord perivascular cells (HUCPVC), which exhibit a higher proliferative rate and frequency than hBM-MSC. Our objective was to establish whether HUCPVC could promote healing of full thickness murine skin defects, and thus find utility as a cell source for dermal repair. To this end, bilateral full thickness defects were created on the dorsum of Balb/c nude mice. Fibrin was used as delivery vehicle for 1 × 106 PKH67-labeled HUCPVC with contralateral controls receiving fibrin only. Epifluorescent and brightfield microscopic evaluation of the wound site was carried out at 3 and 7 days while mechanical testing of wounds was carried out at 3, 7 and 10 days. Our results show that by 3 days, marked contraction of the wound was observed in the fibrin controls whilst the HUCPVC samples exhibited neither collapse nor contraction of the defect, and the dermal repair tissue was considerably thicker and more organized. By 7 days, complete re-epithelialization of the HUCPVC wounds was observed whilst in the controls re-epithelialization was limited to the wound margins. Wound strength was significantly increased in the HUCPVC treatment group by 3 and 7 days but no statistical difference was seen at 10 days. We conclude that HUCPVCs accelerate early wound healing in full thickness skin defects and thus represent a putative source of human MSCs for use in dermal tissue engineering. PMID:21220956

  17. Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

    PubMed Central

    Choi, In Young; Lim, HoTae; Lee, Gabsang

    2014-01-01

    A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases. Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner. PMID:24798302

  18. [Umbilical cord mesenchymal stem cell transplantation for treatment of experimental autoimmune myasthenia gravis in rats].

    PubMed

    Yu, Jing-Xia; Chen, Fang; Sun, Jun; Wang, Ji-Ming; Zhao, Qin-Jun; Ren, Xin-Jun; Ma, Feng-Xia; Yang, Shao-Guang; Han, Zhi-Bo; Han, Zhong-Chao

    2011-06-01

    Umbilical cord mesenchymal stem cell (UCMSC) transplantation has been widely used in the treatment of a variety of diseases due to their advantages such as abundant resources, low immunogenicity and large ex vivo expansion capacity. This study was aimed to investigate the effects of UCMSC on experimental autoimmune myasthenia gravis (EAMG) rats. The distribution of human-derived cells was observed by immunofluorescence method, the effect of MSC on B-cell in situ-secreted antibodies was assayed by ELISPOT, the secreted IFN-γ level was detected by using Transwell test. The results showed that UCMSC were able to migrate to inflammation region and lymph nudes, moreover human-derived cells could be detected in medulla zone of lymph nudes. In vitro in situ detection of AchR specific antibody secretion revealed that the full contact of MSC with lymphnode-derived lymphocytes could effectively inhibit production of AchR antibody. Transwell test indicated that the direct contact of UCMSC with CD4 T cells could effectively decrease production of IFN-γ, which modulated the unbalance between Th1/Th2 to a certain extent. It is concluded that UCMSC can regulate the immune system by direct cell-cell contact or/and release of cytokines, which bring a new insight into knowledge about MSC-based therapy for EAMG. PMID:21729563

  19. Cytogenetic analysis of human somatic cell haploidization.

    PubMed

    Galat, V; Ozen, S; Rechitsky, S; Kuliev, A; Verlinsky, Y

    2005-02-01

    Despite recent interest in the derivation of female and male gametes through somatic cell nuclear transfer, there is still insufficient data on chromosomal analysis of these gametes resulting from haploidization, especially involving a human nuclear donor and recipient oocytes. The objective of this study was to investigate the fidelity of chromosomal separation during haploidization of human cumulus cells by in-vitro matured human enucleated MII oocytes. A total of 129 oocytes were tested 4-7, 8-14, or 15-21 h after nuclear transfer (NT) followed by electro-stimulation, resulting in 71.3% activation efficiency on average. Haploidization was documented by the formation of two separate groups of chromosomes, originating from either polar body/pronucleus (PB/PN), or only 2PN, which were tested by 5-colour FISH, or DNA analysis for copy number of chromosomes 13, 16, 18, 21, 22 and X. Two PN were formed more frequently than PB/PN, irrespective of incubation time. In agreement with recent reports on mouse oocytes, as many as 90.2% of the resulting haploid sets tested showed abnormal chromosome segregation, suggesting unsuitability of the resulting artificial gametes for practical application at the present time. PMID:15823223

  20. Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass.

    PubMed

    Guo, Ge; von Meyenn, Ferdinand; Santos, Fatima; Chen, Yaoyao; Reik, Wolf; Bertone, Paul; Smith, Austin; Nichols, Jennifer

    2016-04-12

    Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. PMID:26947977

  1. Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass

    PubMed Central

    Guo, Ge; von Meyenn, Ferdinand; Santos, Fatima; Chen, Yaoyao; Reik, Wolf; Bertone, Paul; Smith, Austin; Nichols, Jennifer

    2016-01-01

    Summary Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. PMID:26947977

  2. Regulatory roles of B cells in infectious diseases.

    PubMed

    Fillatreau, Simon

    2016-01-01

    B lymphocytes provide essential mechanisms of protection against infectious diseases. The secretion of specific antibodies by long-lived plasma cells is thought to account for the improved resistance afforded by most successful vaccines against pathogens. Accordingly, a goal in vaccine development is to induce potent B cell responses in order to drive the efficient formation of long-lived antibody-secreting cells. However, the roles of activated B cells are complex in infectious diseases. It was recently observed that activated B cells could also negatively regulate host defence mechanisms, both during primary infection and, after vaccination, upon secondary challenge, via mechanisms involving their production of the anti-inflammatory cytokines interleukin (IL)-10 and IL-35. Remarkably, the B cells expressing IL-10 and IL-35 in vivo were distinct subsets of IgMhiCD19+CD138hi antibody-secreting cells. A better understanding of the diverse roles of these distinct antibody-secreting cell subsets in immunity and immunological memory, as well as of the signals controlling their generation, might help the rational development of better prophylactic and therapeutic vaccines. PMID:27586794

  3. Chronic lymphocytic leukemia cells diversify and differentiate in vivo via a nonclassical Th1-dependent, Bcl-6–deficient process

    PubMed Central

    Patten, Piers E.M.; Ferrer, Gerardo; Chen, Shih-Shih; Simone, Rita; Marsilio, Sonia; Yan, Xiao-Jie; Gitto, Zachary; Yuan, Chaohui; Kolitz, Jonathan E.; Barrientos, Jacqueline; Allen, Steven L.; Rai, Kanti R.; MacCarthy, Thomas; Chu, Charles C.; Chiorazzi, Nicholas

    2016-01-01

    Xenografting primary tumor cells allows modeling of the heterogeneous natures of malignant diseases and the influences of the tissue microenvironment. Here, we demonstrate that xenografting primary chronic lymphocytic leukemia (CLL) B lymphocytes with activated autologous T cells into alymphoid mice results in considerable CLL B cell division and sizable T cell expansion. Nevertheless, most/all CD5+CD19+ cells are eventually lost, due in part to differentiation into antibody-secreting plasmablasts/plasma cells. CLL B cell differentiation is associated with isotype class switching and development of new IGHV-D-J mutations and occurs via an activation-induced deaminase-dependent pathway that upregulates IRF4 and Blimp-1 without appreciable levels of the expected Bcl-6. These processes were induced in IGHV-unmutated and IGHV-mutated clones by Th1-polarized T-bet+ T cells, not classical T follicular helper (Tfh) cells. Thus, the block in B cell maturation, defects in T cell action, and absence of antigen-receptor diversification, which are often cardinal characteristics of CLL, are not inherent but imposed by external signals and the microenvironment. Although these activities are not dominant features in human CLL, each occurs in tissue proliferation centers where the mechanisms responsible for clonal evolution operate. Thus, in this setting, CLL B cell diversification and differentiation develop by a nonclassical germinal center–like reaction that might reflect the cell of origin of this leukemia. PMID:27158669

  4. Landscape of transcription in human cells

    PubMed Central

    Djebali, Sarah; Davis, Carrie A.; Merkel, Angelika; Dobin, Alex; Lassmann, Timo; Mortazavi, Ali M.; Tanzer, Andrea; Lagarde, Julien; Lin, Wei; Schlesinger, Felix; Xue, Chenghai; Marinov, Georgi K.; Khatun, Jainab; Williams, Brian A.; Zaleski, Chris; Rozowsky, Joel; Röder, Maik; Kokocinski, Felix; Abdelhamid, Rehab F.; Alioto, Tyler; Antoshechkin, Igor; Baer, Michael T.; Bar, Nadav S.; Batut, Philippe; Bell, Kimberly; Bell, Ian; Chakrabortty, Sudipto; Chen, Xian; Chrast, Jacqueline; Curado, Joao; Derrien, Thomas; Drenkow, Jorg; Dumais, Erica; Dumais, Jacqueline; Duttagupta, Radha; Falconnet, Emilie; Fastuca, Meagan; Fejes-Toth, Kata; Ferreira, Pedro; Foissac, Sylvain; Fullwood, Melissa J.; Gao, Hui; Gonzalez, David; Gordon, Assaf; Gunawardena, Harsha; Howald, Cedric; Jha, Sonali; Johnson, Rory; Kapranov, Philipp; King, Brandon; Kingswood, Colin; Luo, Oscar J.; Park, Eddie; Persaud, Kimberly; Preall, Jonathan B.; Ribeca, Paolo; Risk, Brian; Robyr, Daniel; Sammeth, Michael; Schaffer, Lorian; See, Lei-Hoon; Shahab, Atif; Skancke, Jorgen; Suzuki, Ana Maria; Takahashi, Hazuki; Tilgner, Hagen; Trout, Diane; Walters, Nathalie; Wang, Huaien; Wrobel, John; Yu, Yanbao; Ruan, Xiaoan; Hayashizaki, Yoshihide; Harrow, Jennifer; Gerstein, Mark; Hubbard, Tim; Reymond, Alexandre; Antonarakis, Stylianos E.; Hannon, Gregory; Giddings, Morgan C.; Ruan, Yijun; Wold, Barbara; Carninci, Piero; Guigó, Roderic; Gingeras, Thomas R.

    2013-01-01

    Summary Eukaryotic cells make many types of primary and processed RNAs that are found either in specific sub-cellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic sub-cellular localizations are also poorly understood. Since RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell’s regulatory capabilities are focused on its synthesis, processing, transport, modifications and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations taken together prompt to a redefinition of the concept of a gene. PMID:22955620

  5. Mechanobiology of Human Pluripotent Stem Cells

    PubMed Central

    Earls, Jonathan K.; Jin, Sha

    2013-01-01

    Human pluripotent stem cells (hPSCs) are self-renewing and have the potential to differentiate into any cell type in the body, making them attractive cell sources for applications in tissue engineering and regenerative medicine. However, in order for hPSCs to find use in the clinic, the mechanisms underlying their self-renewal and lineage commitment must be better understood. Many technologies that have been developed for the maintenance and directed differentiation of hPSCs involve the use of soluble growth factors, but recent studies suggest that other elements of the hPSC microenvironment also influence the growth and differentiation of hPSCs. This includes the influences of cell–cell interactions, substrate mechanics, cellular interactions with extracellular matrix, as well as the nanotopography of the substrate and physical forces such as shear stress, cyclic mechanical strain, and compression. In this review, we highlight the recent progress of this area of research and discuss ways in which the mechanical cues may be incorporated into hPSC culture regimes to improve methods for expanding and differentiating hPSCs. PMID:23472616

  6. Innate Immunity in Human Embryonic Stem Cells: Comparison with Adult Human Endothelial Cells

    PubMed Central

    Badiger, Rekha; Paul-Clark, Mark; Moreno, Laura; Lendvai, Zsuzsanna; Wright, Jamie S.; Ali, Nadire N.; Harding, Sian E.; Mitchell, Jane A.

    2010-01-01

    Treatment of human disease with human embryonic stem cell (hESC)-derived cells is now close to reality, but little is known of their responses to physiological and pathological insult. The ability of cells to respond via activation of Toll like receptors (TLR) is critical in innate immune sensing in most tissues, but also extends to more general danger sensing, e.g. of oxidative stress, in cardiomyocytes. We used biomarker release and gene-array analysis to compare responses in hESC before and after differentiation, and to those in primary human endothelial cells. The presence of cardiomyocytes and endothelial cells was confirmed in differentiated cultures by immunostaining, FACS-sorting and, for cardiomyocytes, beating activity. Undifferentiated hESC did not respond with CXCL8 release to Gram positive or Gram negative bacteria, or a range of PAMPs (pathogen associated molecular patterns) for TLRs 1-9 (apart from flagellin, an activator of TLR5). Surprisingly, lack of TLR-dependent responses was maintained over 4 months of differentiation of hESC, in cultures which included cardiomyocytes and endothelial cells. In contrast, primary cultures of human aortic endothelial cells (HAEC) demonstrated responses to a broad range of PAMPs. Expression of downstream TLR signalling pathways was demonstrated in hESC, and IL-1β, TNFα and INFγ, which bypass the TLRs, stimulated CXCL8 release. NFκB pathway expression was also present in hESC and NFκB was able to translocate to the nucleus. Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals. TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin. These findings have potential implications for survival and function of grafted hESC-derived cells. PMID:20463927

  7. Human Pluripotent Stem Cells for Modelling Human Liver Diseases and Cell Therapy

    PubMed Central

    Dianat, Noushin; Steichen, Clara; Vallier, Ludovic; Weber, Anne; Dubart-Kupperschmitt, Anne

    2013-01-01

    The liver is affected by many types of diseases, including metabolic disorders and acute liver failure. Orthotopic liver transplantation (OLT) is currently the only effective treatment for life-threatening liver diseases but transplantation of allogeneic hepatocytes has now become an alternative as it is less invasive than OLT and can be performed repeatedly. However, this approach is hampered by the shortage of organ donors, and the problems related to the isolation of high quality adult hepatocytes, their cryopreservation and their absence of proliferation in culture. Liver is also a key organ to assess the pharmacokinetics and toxicology of xenobiotics and for drug discovery, but appropriate cell culture systems are lacking. All these problems have highlighted the need to explore other sources of cells such as stem cells that could be isolated, expanded to yield sufficiently large populations and then induced to differentiate into functional hepatocytes. The presence of a niche of “facultative” progenitor and stem cells in the normal liver has recently been confirmed but they display no telomerase activity. The recent discovery that human induced pluripotent stem cells can be generated from somatic cells has renewed hopes for regenerative medicine and in vitro disease modelling, as these cells are easily accessible. We review here the present progresses, limits and challenges for the generation of functional hepatocytes from human pluripotent stem cells in view of their potential use in regenerative medicine and drug discovery. PMID:23444872

  8. T helper cell activation and human retroviral pathogenesis.

    PubMed Central

    Copeland, K F; Heeney, J L

    1996-01-01

    T helper (Th) cells are of central importance in regulating many critical immune effector mechanisms. The profile of cytokines produced by Th cells correlates with the type of effector cells induced during the immune response to foreign antigen. Th1 cells induce the cell-mediated immune response, while Th2 cells drive antibody production. Th cells are the preferential targets of human retroviruses. Infections with human T-cell leukemia virus (HTLV) or human immunodeficiency virus (HIV) result in the expansion of Th cells by the action of HTLV (adult T-cell leukemia) or the progressive loss of T cells by the action of HIV (AIDS). Both retrovirus infections impart a high-level activation state in the host immune cells as well as systemically. However, diverging responses to this activation state have contrasting effects on the Th-cell population. In HIV infection, Th-cell loss has been attributed to several mechanisms, including a selective elimination of cells by apoptosis. The induction of apoptosis in HIV infection is complex, with many different pathways able to induce cell death. In contrast, infection of Th cells with HTLV-1 affords the cell a protective advantage against apoptosis. This advantage may allow the cell to escape immune surveillance, providing the opportunity for the development of Th-cell cancer. In this review, we will discuss the impact of Th-cell activation and general immune activation on human retrovirus expression with a focus upon Th-cell function and the progression to disease. PMID:8987361

  9. Studies in human skin epithelial cell carcinogenesis

    SciTech Connect

    Lehman, T.A.

    1987-01-01

    Metabolism and DNA adduct formation of benzo(a)pyrene (BP) by human epidermal keratinocytes pretreated with inhibitors or inducer of cytochrame P450 was studied. To study DNA adduct analysis, cultures were pretreated as described above, and then treated with non-radiolabeled BP. DNA was prepared from these cultures, digested to the nucleotide level, and /sup 32/P-postlabeled for adduct analysis. Cultures pretreated with BHA, 7,8-BF or disulfiralm formed significantly fewer BPDE I-dB adducts than non-pretreated cultures, while cultures pretreated with MeBHA formed more BPDE-I-dG adducts. MeBHA increased BP activation and adduct formation inhuman keratinocyte in cultures by inducing a specific isoenzyme of cytochrome P450 which preferentially increases the oxidative metabolism of BP to 7,8 diol BP and 7,8 diol BP to BPDE I. To approximate an in vivo human system, metabolism of BPDE I by human skin xenografts treated with cell cycles modulators was studied. When treated with BPDE I, specific carcinogen-DNA adducts were formed. Separation and identification of these adducts by the /sup 32/P-postlabeling technique indicated that the 7R- and 7S-BPDE I-dG adducts were the major adducts.

  10. Inhibition of Human Colon Cancer Growth by Antibody-Directed Human LAK Cells in SCID Mice

    NASA Astrophysics Data System (ADS)

    Takahashi, Hiroshi; Nakada, Tetsuya; Puisieux, Isabelle

    1993-03-01

    Advanced human colon cancer does not respond to lymphokine-activated killer (LAK) cells. In order to direct cytotoxic cells to the tumor, human LAK cells linked with antibodies to a tumor cell surface antigen were tested with established hepatic metastases in severe combined immunodeficient (SCID) mice. These cells had increased uptake into the tumor and suppression of tumor growth as compared with LAK cells alone, thereby improving the survival of tumor-bearing mice. Thus, tumor growth can be inhibited by targeted LAK cells, and SCID mice can be used to test the antitumor properties of human effector cells.

  11. The effect of cell phones on human health

    NASA Astrophysics Data System (ADS)

    Abu-Isbeih, Ibrahim N.; Saad, Dina

    2011-10-01

    The effect of cell phone radiation on human health is the subject of recent interest and study, as a result of the enormous increase in cell phone usage throughout the world. Cell phones use electromagnetic radiation in the microwave range, which some believe may be harmful to human health. Other digital wireless systems, such as data communication networks, produce similar radiation. The objective of this survey is to review the effects of cell phones on human health: A large body of research exists, both epidemiological and experimental, in non-human animals and in humans, of which the majority shows no definite causative relationship between exposure to cell phones and harmful biological effects in humans. This is often paraphrased simply as the balance of evidence showing no harm to humans from cell phones, although a significant number of individual studies do suggest such a relationship, or are inconclusive.

  12. Human Naive Embryonic Stem Cells: How Full Is the Glass?

    PubMed

    Wang, Yixuan; Gao, Shaorong

    2016-03-01

    Human naive embryonic stem cells in the ground state of pluripotency provide a new opportunity to study human developmental biology and potential clinical applications. Two studies now report related work in human naive stem cell derivation and DNA methylation analysis, with one reporting some differences from oocyte and blastocyst profiles. PMID:26942847

  13. Trichloroethylene toxicity in a human hepatoma cell line

    SciTech Connect

    Thevenin, E.; McMillian, J.

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  14. Production of monoclonal and polyclonal antibodies against human alphafetoprotein, a hepatocellular tumor marker.

    PubMed

    Chou, Shu-Fen; Hsu, Wen-Lin; Hwang, Jing-Min; Chen, Chien-Yuan

    2002-08-01

    The objective of this study is to produce and purify monoclonal antibodies and polyclonal antibodies (PAbs) against human alphafetoprotein (AFP). Hyperimmune ICR mice produced PAbs after injection with 0.5 mL pristane, and were injected with NS-1 myeloma cells 2 weeks later. Hyperimmune Balb/c mice were used for the production of MAbs. Mice were immunized four times, given a final boost, and their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG 1500. The fused cells were then selected in the hypoxanthine, aminopterine, and thymidine (HAT)-RPMIX medium. Anti-AFP antibody-secreting hybridoma cell lines with high titer were cloned by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in 15% fetal bovine serum (FBS), hypoxanthine, thymidine (HT)-RPMIX medium. Twelve murine hybridoma producing anti-AFP MAbs were obtained and designated as A73F3, A73E8, B73C5, A73G3, A73F8, 67B3, B73C2, B73E1, A73G2, B73G7, B73D7, and B73F4. Isotypes of these MAbs were identified as IgG(1) heavy chain and kappa light chain. The MAbs with high purity were obtained by affinity chromatography. The purity analysis of AFP and the MAbs was performed by capillary electrophoresis. PMID:12193284

  15. Generation of induced pluripotent stem cells from human blood.

    PubMed

    Loh, Yuin-Han; Agarwal, Suneet; Park, In-Hyun; Urbach, Achia; Huo, Hongguang; Heffner, Garrett C; Kim, Kitai; Miller, Justine D; Ng, Kitwa; Daley, George Q

    2009-05-28

    Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage. PMID:19299331

  16. Alternative Sources of Adult Stem Cells: Human Amniotic Membrane

    NASA Astrophysics Data System (ADS)

    Wolbank, Susanne; van Griensven, Martijn; Grillari-Voglauer, Regina; Peterbauer-Scherb, Anja

    Human amniotic membrane is a highly promising cell source for tissue engineering. The cells thereof, human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC), may be immunoprivileged, they represent an early developmental status, and their application is ethically uncontroversial. Cell banking strategies may use freshly isolated cells or involve in vitro expansion to increase cell numbers. Therefore, we have thoroughly characterized the effect of in vitro cultivation on both phenotype and differentiation potential of hAEC. Moreover, we present different strategies to improve expansion including replacement of animal-derived supplements by human platelet products or the introduction of the catalytic subunit of human telomerase to extend the in vitro lifespan of amniotic cells. Characterization of the resulting cultures includes phenotype, growth characteristics, and differentiation potential, as well as immunogenic and immunomodulatory properties.

  17. Derivation and differentiation of haploid human embryonic stem cells.

    PubMed

    Sagi, Ido; Chia, Gloryn; Golan-Lev, Tamar; Peretz, Mordecai; Weissbein, Uri; Sui, Lina; Sauer, Mark V; Yanuka, Ofra; Egli, Dieter; Benvenisty, Nissim

    2016-04-01

    Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development. PMID:26982723

  18. Interleukin 7 independent development of human B cells.

    PubMed Central

    Prieyl, J A; LeBien, T W

    1996-01-01

    Mammalian hematopoietic stem cell (HSC) commitment and differentiation into lymphoid lineage cells proceed through a series of developmentally restricted progenitor compartments. A complete understanding of this process, and how it differs from HSC commitment and differentiation into cells of the myeloid/erythroid lineages, requires the development of model systems that support HSC commitment to the lymphoid lineages. We now describe a human bone marrow stromal cell culture that preferentially supports commitment and differentiation of human HSC to CD19+ B-lineage cells. Fluorescence activated cell sorterpurified CD34++/lineage-cells were isolated from fetal bone marrow and cultured on human fetal bone marrow stromal cells in serum-free conditions containing no exogenous cytokines. Over a period of 3 weeks, CD34++/lineage- cells underwent commitment, differentiation, and expansion into the B lineage. Progressive changes included: loss of CD34, acquisition of and graded increases in the level of cell surface CD19, and appearance of immature B cells expressing mu/kappa or mu/lambda cell surface Ig receptors. The tempo and phenotype of B-cell development was not influenced by the addition of IL-7 (10 ng/ml), or by the addition of goat anti-IL-7 neutralizing antibody. These results indicate a profound difference between mouse and human in the requirement for IL-7 in normal B-cell development, and provide an experimental system to identify and characterize human bone marrow stromal cell-derived molecules crucial for human B lymphopoiesis. PMID:8816803

  19. Data defining markers of human neural stem cell lineage potential.

    PubMed

    Oikari, Lotta E; Okolicsanyi, Rachel K; Griffiths, Lyn R; Haupt, Larisa M

    2016-06-01

    Neural stem cells (NSCs) and neural progenitor cells (NPCs) are self-renewing and multipotent cells, however, NPCs are considered to be more lineage-restricted with a reduced self-renewing capacity. We present data comparing the expression of 21 markers encompassing pluripotency, self-renewal (NSC) as well as neuronal and glial (astrocyte and oligodendrocyte) lineage specification and 28 extracellular proteoglycan (PG) genes and their regulatory enzymes between embryonic stem cell (ESC)-derived human NSCs (hNSC H9 cells, Thermo Fisher) and human cortex-derived normal human NPCs (nhNPCs, Lonza). The data demonstrates expression differences of multiple lineage and proteoglycan-associated genes between hNSC H9 cells and nhNPCs. Data interpretation of markers and proteoglycans defining NSC and neural cell lineage characterisation can be found in "Cell surface heparan sulfate proteoglycans as novel markers of human neural stem cell fate determination" (Oikari et al. 2015) [1]. PMID:26958640

  20. Data defining markers of human neural stem cell lineage potential

    PubMed Central

    Oikari, Lotta E.; Okolicsanyi, Rachel K.; Griffiths, Lyn R.; Haupt, Larisa M.

    2016-01-01

    Neural stem cells (NSCs) and neural progenitor cells (NPCs) are self-renewing and multipotent cells, however, NPCs are considered to be more lineage-restricted with a reduced self-renewing capacity. We present data comparing the expression of 21 markers encompassing pluripotency, self-renewal (NSC) as well as neuronal and glial (astrocyte and oligodendrocyte) lineage specification and 28 extracellular proteoglycan (PG) genes and their regulatory enzymes between embryonic stem cell (ESC)-derived human NSCs (hNSC H9 cells, Thermo Fisher) and human cortex-derived normal human NPCs (nhNPCs, Lonza). The data demonstrates expression differences of multiple lineage and proteoglycan-associated genes between hNSC H9 cells and nhNPCs. Data interpretation of markers and proteoglycans defining NSC and neural cell lineage characterisation can be found in “Cell surface heparan sulfate proteoglycans as novel markers of human neural stem cell fate determination” (Oikari et al. 2015) [1]. PMID:26958640

  1. FOXP3+ regulatory T cells in the human immune system.

    PubMed

    Sakaguchi, Shimon; Miyara, Makoto; Costantino, Cristina M; Hafler, David A

    2010-07-01

    Forkhead box P3 (FOXP3)(+) regulatory T (T(Reg)) cells are potent mediators of dominant self tolerance in the periphery. But confusion as to the identity, stability and suppressive function of human T(Reg) cells has, to date, impeded the general therapeutic use of these cells. Recent studies have suggested that human T(Reg) cells are functionally and phenotypically diverse. Here we discuss recent findings regarding human T(Reg) cells, including the ontogeny and development of T(Reg) cell subsets that have naive or memory phenotypes, the unique mechanisms of suppression mediated by T(Reg) cell subsets and factors that regulate T(Reg) cell lineage commitment. We discuss future studies that are needed for the successful therapeutic use of human T(Reg) cells. PMID:20559327

  2. Human Pancreatic β-Cell G1/S Molecule Cell Cycle Atlas

    PubMed Central

    Fiaschi-Taesch, Nathalie M.; Kleinberger, Jeffrey W.; Salim, Fatimah G.; Troxell, Ronnie; Wills, Rachel; Tanwir, Mansoor; Casinelli, Gabriella; Cox, Amy E.; Takane, Karen K.; Scott, Donald K.; Stewart, Andrew F.

    2013-01-01

    Expansion of pancreatic β-cells is a key goal of diabetes research, yet induction of adult human β-cell replication has proven frustratingly difficult. In part, this reflects a lack of understanding of cell cycle control in the human β-cell. Here, we provide a comprehensive immunocytochemical “atlas” of G1/S control molecules in the human β-cell. This atlas reveals that the majority of these molecules, previously known to be present in islets, are actually present in the β-cell. More importantly, and in contrast to anticipated results, the human β-cell G1/S atlas reveals that almost all of the critical G1/S cell cycle control molecules are located in the cytoplasm of the quiescent human β-cell. Indeed, the only nuclear G1/S molecules are the cell cycle inhibitors, pRb, p57, and variably, p21: none of the cyclins or cdks necessary to drive human β-cell proliferation are present in the nuclear compartment. This observation may provide an explanation for the refractoriness of human β-cells to proliferation. Thus, in addition to known obstacles to human β-cell proliferation, restriction of G1/S molecules to the cytoplasm of the human β-cell represents an unanticipated obstacle to therapeutic human β-cell expansion. PMID:23493570

  3. Direct reprogramming of human neural stem cells by OCT4.

    PubMed

    Kim, Jeong Beom; Greber, Boris; Araúzo-Bravo, Marcos J; Meyer, Johann; Park, Kook In; Zaehres, Holm; Schöler, Hans R

    2009-10-01

    Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells. PMID:19718018

  4. Reversine Induced Multinucleated Cells, Cell Apoptosis and Autophagy in Human Non-Small Cell Lung Cancer Cells

    PubMed Central

    Lin, Ching-Yen; Chen, Yih-Yuan; Chen, Ping-Tzu; Tseng, Ya-Shih

    2016-01-01

    Reversine, an A3 adenosine receptor antagonist, has been shown to induce differentiated myogenic-lineage committed cells to become multipotent mesenchymal progenitor cells. We and others have reported that reversine has an effect on human tumor suppression. This study revealed anti-tumor effects of reversine on proliferation, apoptosis and autophagy induction in human non-small cell lung cancer cells. Treatment of these cells with reversine suppressed cell growth in a time- and dosage-dependent manner. Moreover, polyploidy occurred after reversine treatment. In addition, caspase-dependent apoptosis and activation of autophagy by reversine in a dosage-dependent manner were also observed. We demonstrated in this study that reversine contributes to growth inhibition, apoptosis and autophagy induction in human lung cancer cells. Therefore, reversine used as a potential therapeutic agent for human lung cancer is worthy of further investigation. PMID:27385117

  5. Development and function of human innate immune cells in a humanized mouse model

    PubMed Central

    Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V.; Teichmann, Lino L.; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A. Karolina; Manz, Markus G.; Flavell, Richard A.

    2014-01-01

    Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models are unable to support development of human innate immune cells, including myeloid cells and NK cells. Here we describe a mouse strain, called MI(S)TRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked in to their respective mouse loci. The human cytokines support the development and function of monocytes/macrophages and natural killer cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MI(S)TRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology. PMID:24633240

  6. Generation of Human iNKT Cell Lines

    PubMed Central

    Li, Xiangming; Tsuji, Moriya; Schneck, Jonathan; Webb, Tonya J.

    2016-01-01

    Natural killer T (NKT) cells comprise an important immunoregulatory T cell subset and express cell surface proteins characteristic of both natural killer cells and T cells. Invariant NKT (iNKT) cells are activated by lipid antigen presented in the context of CD1d molecules, in contrast to classic T cell subsets, which recognize peptide antigens presented by MHC molecules. Following activation, iNKT cells rapidly secrete large amounts of cytokines and can lyse tumor cells and virally infected cells; however, iNKT cells are reduced in patients with autoimmune disease and cancer. The potential to characterize and investigate the prospective use of iNKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human iNKT cells. In this protocol, we describe a method to generate and propagate primary human iNKT cells. Specifically, primary iNKT cells were isolated from human peripheral blood mononuclear cells (PBMC), and then expanded periodically with irradiated α-GalCer loaded autologous immature dendritic cells (DC) in the presence of human IL-2.

  7. Development and characterization of a new human hepatic cell line.

    PubMed

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  8. Development and characterization of a new human hepatic cell line

    PubMed Central

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  9. Evidence for bystander signalling between human trophoblast cells and human embryonic stem cells.

    PubMed

    Jones, Anna J; Gokhale, Paul J; Allison, Thomas F; Sampson, Barry; Athwal, Sharan; Grant, Simon; Andrews, Peter W; Allen, Nicholas D; Case, C Patrick

    2015-01-01

    Maternal exposure during pregnancy to toxins can occasionally lead to miscarriage and malformation. It is currently thought that toxins pass through the placental barrier, albeit bi-layered in the first trimester, and damage the fetus directly, albeit at low concentration. Here we examined the responses of human embryonic stem (hES) cells in tissue culture to two metals at low concentration. We compared direct exposures with indirect exposures across a bi-layered model of the placenta cell barrier. Direct exposure caused increased DNA damage without apoptosis or a loss of cell number but with some evidence of altered differentiation. Indirect exposure caused increased DNA damage and apoptosis but without loss of pluripotency. This was not caused by metal ions passing through the barrier. Instead the hES cells responded to signalling molecules (including TNF-α) secreted by the barrier cells. This mechanism was dependent on connexin 43 mediated intercellular 'bystander signalling' both within and between the trophoblast barrier and the hES colonies. These results highlight key differences between direct and indirect exposure of hES cells across a trophoblast barrier to metal toxins. It offers a theoretical possibility that an indirectly mediated toxicity of hES cells might have biological relevance to fetal development. PMID:26170169

  10. Evidence for bystander signalling between human trophoblast cells and human embryonic stem cells

    PubMed Central

    Jones, Anna J; Gokhale, Paul J; Allison, Thomas F; Sampson, Barry; Athwal, Sharan; Grant, Simon; Andrews, Peter W; Allen, Nicholas D; Patrick Case, C

    2015-01-01

    Maternal exposure during pregnancy to toxins can occasionally lead to miscarriage and malformation. It is currently thought that toxins pass through the placental barrier, albeit bi-layered in the first trimester, and damage the fetus directly, albeit at low concentration. Here we examined the responses of human embryonic stem (hES) cells in tissue culture to two metals at low concentration. We compared direct exposures with indirect exposures across a bi-layered model of the placenta cell barrier. Direct exposure caused increased DNA damage without apoptosis or a loss of cell number but with some evidence of altered differentiation. Indirect exposure caused increased DNA damage and apoptosis but without loss of pluripotency. This was not caused by metal ions passing through the barrier. Instead the hES cells responded to signalling molecules (including TNF-α) secreted by the barrier cells. This mechanism was dependent on connexin 43 mediated intercellular ‘bystander signalling’ both within and between the trophoblast barrier and the hES colonies. These results highlight key differences between direct and indirect exposure of hES cells across a trophoblast barrier to metal toxins. It offers a theoretical possibility that an indirectly mediated toxicity of hES cells might have biological relevance to fetal development. PMID:26170169

  11. Effects of Human Umbilical Cord Mesenchymal Stem Cells on Human Trophoblast Cell Functions In Vitro

    PubMed Central

    Huang, Yajing; Wu, Yanming; Chang, Xinwen; Li, Yan; Wang, Kai; Duan, Tao

    2016-01-01

    Trophoblast cell dysfunction is involved in many disorders during pregnancy such as preeclampsia and intrauterine growth restriction. Few treatments exist, however, that target improving trophoblast cell function. Human umbilical cord mesenchymal stem cells (hUCMSCs) are capable of self-renewing, can undergo multilineage differentiation, and have homing abilities; in addition, they have immunomodulatory effects and paracrine properties and thus are a prospective source for cell therapy. To identify whether hUCMSCs can regulate trophoblast cell functions, we treated trophoblast cells with hUCMSC supernatant or cocultured them with hUCMSCs. Both treatments remarkably enhanced the migration and invasion abilities of trophoblast cells and upregulated their proliferation ability. At a certain concentration, hUCMSCs also modulated hCG, PIGF, and sEndoglin levels in the trophoblast culture medium. Thus, hUCMSCs have a positive effect on trophoblast cellular functions, which may provide a new avenue for treatment of placenta-related diseases during pregnancy. PMID:26949402

  12. Eliminating malignant contamination from therapeutic human spermatogonial stem cells

    PubMed Central

    Dovey, Serena L.; Valli, Hanna; Hermann, Brian P.; Sukhwani, Meena; Donohue, Julia; Castro, Carlos A.; Chu, Tianjiao; Sanfilippo, Joseph S.; Orwig, Kyle E.

    2013-01-01

    Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4–contaminated human testicular cell suspensions was performed to isolate EpCAM+/HLA-ABC–/CD49e– (putative spermatogonia) and EpCAM–/HLA-ABC+/CD49e+ (putative MOLT-4) cell fractions. The EpCAM+/HLA-ABC–/CD49e– fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to–nude mouse xenotransplantation. The EpCAM–/HLA-ABC+/CD49e+ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression. PMID:23549087

  13. Human Wharton's jelly stem cells, its conditioned medium and cell-free lysate inhibit the growth of human lymphoma cells.

    PubMed

    Lin, Hao Daniel; Fong, Chui Yee; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

    2014-08-01

    Several groups have reported that primitive mesenchymal stem cells from the gelatinous matrix of the Wharton's jelly of the human umbilical cord (hWJSCs) possess tumoricidal properties and inhibit the growth of solid tumours such as human mammary carcinoma, ovarian carcinoma and osteosarcoma. This unique characteristic led to the hypothesis that hWJSCs serve as a natural defence against migrating cancer cells from mother to fetus thus explaining why tumorigenesis in the fetus is rare. However, it is not known whether non-solid malignant hematopoietic cells are also inhibited by hWJSCs and what the exact tumoricidal mechanisms are. We therefore evaluated the influence of hWJSCs and its extracts on Burkitt's lymphoma cells. Cell proliferation (BrdU and Ki67+), viability (MTT) and cell death (Annexin V-Propidium iodide and live/dead) assays showed significant inhibition of lymphoma cell growth after 48 h exposure to hWJSCs or its extracts compared to controls. Increased cell death was observed at sub-G1 and S and decreased proliferation at G2/M phases of the mitotic cycle. Superoxide dismutase and hydrogen peroxide activity were significantly increased and glutathione peroxidase significantly decreased in treated lymphoma cells. Time lapse imaging and confocal z-stack images showed yellow fluorescent in situ hybridization (FISH) signals of lymphoma cell Y chromosomes within the cytoplasm of female red labelled hWJSCs. We hypothesize that the growth of lymphoma cells is inhibited by the molecules secreted by hWJSCs that use oxidative stress pathways to induce cell death followed by engulfment of the apoptotic remains of the lymphoma cells by the hWJSCs. PMID:24789672

  14. Stem Cells: A Renaissance in Human Biology Research.

    PubMed

    Wu, Jun; Izpisua Belmonte, Juan Carlos

    2016-06-16

    The understanding of human biology and how it relates to that of other species represents an ancient quest. Limited access to human material, particularly during early development, has restricted researchers to only scratching the surface of this inherently challenging subject. Recent technological innovations, such as single cell "omics" and human stem cell derivation, have now greatly accelerated our ability to gain insights into uniquely human biology. The opportunities afforded to delve molecularly into scarce material and to model human embryogenesis and pathophysiological processes are leading to new insights of human development and are changing our understanding of disease and choice of therapy options. PMID:27315475

  15. Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions

    PubMed Central

    Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min

    2016-01-01

    Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies. PMID:27294211

  16. Neoplastic human embryonic stem cells as a model of radiation resistance of human cancer stem cells

    PubMed Central

    Dingwall, Steve; Lee, Jung Bok; Guezguez, Borhane; Fiebig, Aline; McNicol, Jamie; Boreham, Douglas; Collins, Tony J.; Bhatia, Mick

    2015-01-01

    Studies have implicated that a small sub-population of cells within a tumour, termed cancer stem cells (CSCs), have an enhanced capacity for tumour formation in multiple cancers and may be responsible for recurrence of the disease after treatment, including radiation. Although comparisons have been made between CSCs and bulk-tumour, the more important comparison with respect to therapy is between tumour-sustaining CSC versus normal stem cells that maintain the healthy tissue. However, the absence of normal known counterparts for many CSCs has made it difficult to compare the radiation responses of CSCs with the normal stem cells required for post-radiotherapy tissue regeneration and the maintenance of tissue homeostasis. Here we demonstrate that transformed human embryonic stem cells (t-hESCs), showing features of neoplastic progression produce tumours resistant to radiation relative to their normal counterpart upon injection into immune compromised mice. We reveal that t-hESCs have a reduced capacity for radiation induced cell death via apoptosis and exhibit altered cell cycle arrest relative to hESCs in vitro. t-hESCs have an increased expression of BclXL in comparison to their normal counterparts and re-sensitization of t-hESCs to radiation upon addition of BH3-only mimetic ABT737, suggesting that overexpression of BclXL underpins t-hESC radiation insensitivity. Using this novel discovery platform to investigate radiation resistance in human CSCs, our study indicates that chemotherapy targeting Bcl2-family members may prove to be an adjuvant to radiotherapy capable of targeting CSCs. PMID:26082437

  17. Comparative mutagenesis of human cells in vivo and in vitro

    SciTech Connect

    Thilly, W.G.

    1992-05-01

    This report discusses measuring methods of point mutations; high density cell cultures for low dose studies; measurement and sequence determination of mutations in DNA; the mutational spectra of styrene oxide and ethlyene oxide in TK-6 cells; mutational spectrum of Cr in human lymphoblast cells; mutational spectra of radon in TK-6 cells; and the mutational spectra of smokeless tobacco. (CBS)

  18. Closing in on Mass Production of Mature Human Beta Cells.

    PubMed

    Kieffer, Timothy J

    2016-06-01

    Human pluripotent stem cell differentiation protocols based on mimicking developmental pathways are getting close to generating fully fledged pancreatic endocrine cells, including insulin-producing beta cells. However, challenges remain in identifying pathways to trigger the attainment of robust glucose responsiveness that occurs postnatally in beta cells. PMID:27257758

  19. The Evolution of Human Cells in Terms of Protein Innovation

    PubMed Central

    Sardar, Adam J.; Oates, Matt E.; Fang, Hai; Forrest, Alistair R.R.; Kawaji, Hideya; Gough, Julian; Rackham, Owen J.L.

    2014-01-01

    Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type–specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type–specific domain architectures. PMID:24692656

  20. Generation of functional hepatocytes from human spermatogonial stem cells.

    PubMed

    Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

    2016-02-23

    To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration. PMID:26840458

  1. Generation of functional hepatocytes from human spermatogonial stem cells

    PubMed Central

    Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

    2016-01-01

    To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration. PMID:26840458

  2. Development of human epithelial cell systems for radiation risk assessment

    NASA Technical Reports Server (NTRS)

    Yang, C. H.; Craise, L. M.

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  3. Human Immunodeficiency Syndromes Affecting Human Natural Killer Cell Cytolytic Activity

    PubMed Central

    Ham, Hyoungjun; Billadeau, Daniel D.

    2013-01-01

    Natural killer (NK) cells are lymphocytes of the innate immune system that secrete cytokines upon activation and mediate the killing of tumor cells and virus-infected cells, especially those that escape the adaptive T cell response caused by the down regulation of MHC-I. The induction of cytotoxicity requires that NK cells contact target cells through adhesion receptors, and initiate activation signaling leading to increased adhesion and accumulation of F-actin at the NK cell cytotoxic synapse. Concurrently, lytic granules undergo minus-end directed movement and accumulate at the microtubule-organizing center through the interaction with microtubule motor proteins, followed by polarization of the lethal cargo toward the target cell. Ultimately, myosin-dependent movement of the lytic granules toward the NK cell plasma membrane through F-actin channels, along with soluble N-ethylmaleimide-sensitive factor attachment protein receptor-dependent fusion, promotes the release of the lytic granule contents into the cleft between the NK cell and target cell resulting in target cell killing. Herein, we will discuss several disease-causing mutations in primary immunodeficiency syndromes and how they impact NK cell-mediated killing by disrupting distinct steps of this tightly regulated process. PMID:24478771

  4. Human liver endothelial cells, but not macrovascular or microvascular endothelial cells, engraft in the mouse liver.

    PubMed

    Filali, Ebtisam El; Hiralall, Johan K; van Veen, Henk A; Stolz, Donna B; Seppen, Jurgen

    2013-01-01

    Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and repopulation capacity of human endothelial cells derived from various tissues. Human endothelial cells were isolated from adult and fetal livers using anti-human CD31 antibody-conjugated magnetic beads. Human macrovascular endothelial cells were obtained from umbilical vein. Human microvascular endothelial cells were isolated from adipose tissue. Cells were characterized using flow cytometry. Liver engraftment and repopulation of endothelial cells was studied after intrasplenic transplantation in monocrotaline-treated immunodeficient mice. Following transplantation, human liver endothelial cells engrafted throughout the mouse liver. With immunoscanning electron microscopy, fenestrae in engrafted human liver endothelial cells were identified, a characteristic feature of liver sinusoidal endothelial cells. In contrast, CD31-negative liver cells, human macrovascular and microvascular endothelial cells were not capable of repopulating mouse liver. Characterization of human liver, macrovascular, and microvascular endothelial cells demonstrated expression of CD31, CD34, and CD146 but not CD45. Our study shows that only human liver endothelial cells, but not macro- and microvascular endothelial cells, have the unique capacity to engraft and repopulate the mouse liver. These results indicate that mature endothelial cells cannot transdifferentiate in vivo and thus do not exhibit phenotypic plasticity. Our results have set a basis for further research to the potential of human liver endothelial cells in liver-directed cell and gene therapy. PMID:23044355

  5. Activation of GPR119 Stimulates Human β-Cell Replication and Neogenesis in Humanized Mice with Functional Human Islets

    PubMed Central

    Ansarullah; Free, Colette; Christopherson, Jenica; Chen, Quanhai; Gao, Jie; Liu, Chengyang; Naji, Ali; Rabinovitch, Alex; Guo, Zhiguang

    2016-01-01

    Using humanized mice with functional human islets, we investigated whether activating GPR119 by PSN632408, a small molecular agonist, can stimulate human β-cell regeneration in vivo. Human islets were transplanted under the left kidney capsule of immunodeficient mice with streptozotocin- (STZ-) induced diabetes. The recipient mice were treated with PSN632408 or vehicle and BrdU daily. Human islet graft function in the mice was evaluated by nonfasting glucose levels, oral glucose tolerance, and removal of the grafts. Immunostaining for insulin, glucagon, and BrdU or Ki67 was performed in islet grafts to evaluate α- and β-cell replication. Insulin and CK19 immunostaining was performed to evaluate β-cell neogenesis. Four weeks after human islet transplantation, 71% of PSN632408-treated mice achieved normoglycaemia compared with 24% of vehicle-treated mice. Also, oral glucose tolerance was significantly improved in the PSN632408-treated mice. PSN632408 treatment significantly increased both human α- and β-cell areas in islet grafts and stimulated α- and β-cell replication. In addition, β-cell neogenesis was induced from pancreatic duct cells in the islet grafts. Our results demonstrated that activation of GPR119 increases β-cell mass by stimulating human β-cell replication and neogenesis. Therefore, GPR119 activators may qualify as therapeutic agents to increase human β-cell mass in patients with diabetes. PMID:27413754

  6. Activin A programs the differentiation of human TFH cells.

    PubMed

    Locci, Michela; Wu, Jennifer E; Arumemi, Fortuna; Mikulski, Zbigniew; Dahlberg, Carol; Miller, Andrew T; Crotty, Shane

    2016-08-01

    Follicular helper T cells (TFH cells) are CD4(+) T cells specialized in helping B cells and are associated both with protective antibody responses and autoimmune diseases. The promise of targeting TFH cells therapeutically has been limited by fragmentary understanding of extrinsic signals that regulate the differentiation of human TFH cells. A screen of a human protein library identified activin A as a potent regulator of TFH cell differentiation. Activin A orchestrated the expression of multiple genes associated with the TFH program, independently or in concert with additional signals. TFH cell programming by activin A was antagonized by the cytokine IL-2. Activin A's ability to drive TFH cell differentiation in vitro was conserved in non-human primates but not in mice. Finally, activin-A-induced TFH programming was dependent on signaling via SMAD2 and SMAD3 and was blocked by pharmacological inhibitors. PMID:27376469

  7. Phenotypic variability in human skin mast cells.

    PubMed

    Babina, Magda; Guhl, Sven; Artuc, Metin; Trivedi, Neil N; Zuberbier, Torsten

    2016-06-01

    Mast cells (MCs) are unique constituents of the human body. While inter-individual differences may influence the ways by which MCs operate in their skin habitat, they have not been surveyed in a comprehensive manner so far. We therefore set out to quantify skin MC variability in a large cohort of subjects. Pathophysiologically relevant key features were quantified and correlated: transcripts of c-kit, FcεRIα, FcεRIβ, FcεRIγ, histidine decarboxylase, tryptase, and chymase; surface expression of c-Kit, FcεRIα; activity of tryptase, and chymase; histamine content and release triggered by FcεRI and Ca(2+) ionophore. While there was substantial variability among subjects, it strongly depended on the feature under study (coefficient of variation 33-386%). Surface expression of FcεRI was positively associated with FcεRIα mRNA content, histamine content with HDC mRNA, and chymase activity with chymase mRNA. Also, MC signature genes were co-regulated in distinct patterns. Intriguingly, histamine levels were positively linked to tryptase and chymase activity, whereas tryptase and chymase activity appeared to be uncorrelated. FcεRI triggered histamine release was highly variable and was unrelated to FcεRI expression but unexpectedly tightly correlated with histamine release elicited by Ca(2+) ionophore. This most comprehensive and systematic work of its kind provides not only detailed insights into inter-individual variability in MCs, but also uncovers unexpected patterns of co-regulation among signature attributes of the lineage. Differences in MCs among humans may well underlie clinical responses in settings of allergic reactions and complex skin disorders alike. PMID:26706922

  8. Analysis of lead toxicity in human cells

    PubMed Central

    2012-01-01

    Background Lead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity. Results We analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were altered in subjects with high

  9. AID-Induced Genotoxic Stress Promotes B Cell Differentiation in the Germinal Center via ATM and LKB1 Signaling

    PubMed Central

    Sherman, Mara H.; Kuraishy, Ali I.; Deshpande, Chetan; Hong, Jason S.; Cacalano, Nicholas A.; Gatti, Richard A.; Manis, John P.; Damore, Michael A.; Pellegrini, Matteo; Teitell, Michael A.

    2010-01-01

    SUMMARY During an immune response, B cells undergo rapid proliferation and AID-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here we report that exogenous- and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patient samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress. PMID:20864035

  10. Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy.

    PubMed

    Bhoj, Vijay G; Arhontoulis, Dimitrios; Wertheim, Gerald; Capobianchi, James; Callahan, Colleen A; Ellebrecht, Christoph T; Obstfeld, Amrom E; Lacey, Simon F; Melenhorst, Jan J; Nazimuddin, Farzana; Hwang, Wei-Ting; Maude, Shannon L; Wasik, Mariusz A; Bagg, Adam; Schuster, Stephen; Feldman, Michael D; Porter, David L; Grupp, Stephen A; June, Carl H; Milone, Michael C

    2016-07-21

    The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity. PMID:27166358

  11. A Cell-Based Approach to the Human Proteome Project

    NASA Astrophysics Data System (ADS)

    Kelleher, Neil L.

    2012-10-01

    The general scope of a project to determine the protein molecules that comprise the cells within the human body is framed. By focusing on protein primary structure as expressed in specific cell types, this concept for a cell-based version of the Human Proteome Project (CB-HPP) is crafted in a manner analogous to the Human Genome Project while recognizing that cells provide a primary context in which to define a proteome. Several activities flow from this articulation of the HPP, which enables the definition of clear milestones and deliverables. The CB-HPP highlights major gaps in our knowledge regarding cell heterogeneity and protein isoforms, and calls for development of technology that is capable of defining all human cell types and their proteomes. The main activities will involve mapping and sorting cell types combined with the application of beyond the state-of-the art in protein mass spectrometry.

  12. Establishment, characterization, and successful adaptive therapy against human tumors of NKG cell, a new human NK cell line.

    PubMed

    Cheng, Min; Ma, Juan; Chen, Yongyan; Zhang, Jianhua; Zhao, Weidong; Zhang, Jian; Wei, Haiming; Ling, Bin; Sun, Rui; Tian, Zhigang

    2011-01-01

    Natural killer (NK) cells play important roles in adoptive cellular immunotherapy against certain human cancers. This study aims to establish a new human NK cell line and to study its role for adoptive cancer immunotherapy. Peripheral blood samples were collected from 54 patients to establish the NK cell line. A new human NK cell line, termed as NKG, was established from a Chinese male patient with rapidly progressive non-Hodgkin's lymphoma. NKG cells showed LGL morphology and were phenotypically identified as CD56(bright) NK cell with CD16(-), CD27(-), CD3(-), αβTCR(-), γδTCR(-), CD4(-), CD8(-), CD19(-), CD161(-), CD45(+), CXCR4(+), CCR7(+), CXCR1(-), and CX3CR1(-). NKG cells showed high expression of adhesive molecules (CD2, CD58, CD11a, CD54, CD11b, CD11c), an array of activating receptors (NKp30, NKp44, NKp46, NKG2D, NKG2C), and cytolysis-related receptors and molecules (TRAIL, FasL, granzyme B, perforin, IFN-γ). The cytotoxicity of NKG cells against tumor cells was higher than that of the established NK cell lines NK-92, NKL, and YT. NKG cell cytotoxicity depended on the presence of NKG2D and NKp30. When irradiated with 8 Gy, NKG cells were still with high cytotoxicity and activity in vitro and with safety in vivo, but without proliferation. Further, the irradiated NKG cells exhibited strong cytotoxicity against human primary ovarian cancer cells in vitro, and against human ovarian cancer in a mouse xenograft model. The adoptive transfer of NKG cells significantly inhibited the ovarian tumor growth, decreased the mortality rate and prolonged the survival, even in cases of advanced diseases. A number of NKG cells were detected in the ovarian tumor tissues during cell therapy. In use of the new human NK cell line, NKG would a promising cellular candidate for adoptive immunotherapy of human cancer. PMID:21669033

  13. Nucleosome Organization in Human Embryonic Stem Cells

    PubMed Central

    Taylor, Jared F.; Khattab, Omar S.; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E.; Wang, Ping H.

    2015-01-01

    The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a “ground state” of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this “ground state” by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of

  14. Delivery of iron to human cells by bovine transferrin. Implications for the growth of human cells in vitro.

    PubMed Central

    Young, S P; Garner, C

    1990-01-01

    Following suggestions that transferrin present in fetal-bovine serum, a common supplement used in tissue-culture media, may not bind well to human cells, we have isolated the protein and investigated its interaction with both human and bovine cells. Bovine transferrin bound to a human cell line, K562, at 4 degrees C with a kd of 590 nM, whereas human transferrin bound with a kd of 3.57 nM, a 165-fold difference. With a bovine cell line, NBL4, bovine transferrin bound with the higher affinity, kd 9.09 nM, whereas human transferrin bound with a kd of 41.7 nM, only a 5-fold difference. These values were reflected in an 8.6-fold difference in the rate of iron delivery by the two proteins to human cells, whereas delivery to bovine cells was the same. Nevertheless, the bovine transferrin was taken up by the human cells by a specific receptor-mediated process. Human cells cultured in bovine diferric transferrin at 40 micrograms/ml, the concentration expected in the presence of 10% fetal-bovine serum, failed to thrive, whereas cells cultured in the presence of human transferrin proliferated normally. These results suggest that growth of human cells in bovine serum could give rise to a cellular iron deficiency, which may in turn lead to the selection of clones of cells adapted for survival with less iron. This has important consequences for the use of such cells as models, since they may have aberrant iron-dependent pathways and perhaps other unknown alterations in cell function. PMID:2302189

  15. Development of human B cells and antibodies following human hematopoietic stem cell transplantation to Rag2(-/-)γc(-/-) mice.

    PubMed

    Tanner, Anne; Hallam, Steven J; Nielsen, Stanton J; Cuadra, German I; Berges, Bradford K

    2015-06-01

    Humanized mice represent a valuable model system to study the development and functionality of the human immune system. In the RAG-hu mouse model highly immunodeficient Rag2(-/-)γc(-/-) mice are transplanted with human CD34(+) hematopoietic stem cells, resulting in human hematopoiesis and a predominant production of B and T lymphocytes. Human adaptive immune responses have been detected towards a variety of antigens in humanized mice but both cellular and humoral immune responses tend to be weak and sporadically detected. The underlying mechanisms for inconsistent responses are poorly understood. Here, we analyzed the kinetics of human B cell development and antibody production in RAG-hu mice to better understand the lack of effective antibody responses. We found that T cell levels in blood did not significantly change from 8 to 28 weeks post-engraftment, while B cells reached a peak at 14 weeks. Concentrations of 3 antibody classes (IgM, IgG, IgA) were found to be at levels about 0.1% or less of normal human levels, but human antibodies were still detected up to 32 weeks after engraftment. Human IgM was detected in 92.5% of animals while IgG and IgA were detected in about half of animals. We performed flow cytometric analysis of human B cells in bone marrow, spleen, and blood to examine the presence of precursor B cells, immature B cells, naïve B cells, and plasma B cells. We detected high levels of surface IgM(+) B cells (immature and naïve B cells) and low levels of plasma B cells in these organs, suggesting that B cells do not mature properly in this model. Low levels of human T cells in the spleen were observed, and we suggest that the lack of T cell help may explain poor B cell development and antibody responses. We conclude that human B cells that develop in humanized mice do not receive the signals necessary to undergo class-switching or to secrete antibody effectively, and we discuss strategies to potentially overcome these barriers. PMID:25843523

  16. Human embryonic stem cell differentiation toward regional specific neural precursors.

    PubMed

    Erceg, Slaven; Ronaghi, Mohammad; Stojković, Miodrag

    2009-01-01

    Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that have the capacity to differentiate into a wide variety of cell types. This potentiality represents a promising source to overcome many human diseases by providing an unlimited supply of all cell types, including cells with neural characteristics. Therefore, this review summarizes early neural development and the potential of hESCs to differentiate under in vitro conditions, examining at the same time the potential use of differentiated hESCs for therapeutic applications for neural tissue and cell regeneration. PMID:18845761

  17. Human Embryonic Stem Cell Differentiation Toward Regional Specific Neural Precursors

    PubMed Central

    Erceg, Slaven; Ronaghi, Mohammad; Stojković, Miodrag

    2009-01-01

    Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that have the capacity to differentiate into a wide variety of cell types. This potentiality represents a promising source to overcome many human diseases by providing an unlimited supply of all cell types, including cells with neural characteristics. Therefore, this review summarizes early neural development and the potential of hESCs to differentiate under in vitro conditions, examining at the same time the potential use of differentiated hESCs for therapeutic applications for neural tissue and cell regeneration. PMID:18845761

  18. Transformation of human cells by DNAs ineffective in transformation of NIH 3T3 cells

    SciTech Connect

    Sutherland, B.M.; Bennett, P.B.; Freeman, A.G.; Moore, S.P.; Strickland, P.T.

    1985-04-01

    Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.

  19. Isolation and in vitro differentiation of human erythroid precursor cells.

    PubMed

    Kim, H C; Marks, P A; Rifking, R A; Maniatis, G M; Bank, A

    1976-05-01

    There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia. PMID:1260133

  20. Cultures of mast cell-like (MCL) cells from human pleural exudate cells.

    PubMed

    Krüger, G; Sterry, W; Czarnetzki, B M

    1983-03-01

    Under special culture conditions, rat peritoneal macrophages have previously been shown to transform into mast cells. This method has been adapted here to the human species. Adherent large mononuclear cells from human pleural exudates were cultured in a medium supplemented with horse serum (30%) and fibroblast supernatants (30%). Metachromatic staining (toluidine blue, pH 3.6) of cytoplasmic granules appeared first in a small percentage of cells by days 5-6 of culture and reached a high intensity in 50% of the cells between days 12-22. Histamine levels within the cells increased by a factor of 7 during this same time period and the cell size by a factor of 3. Cultures could be maintained for about three weeks, since viability and total cell number decreased on extended culture. The data suggest that mononuclear cells in inflammatory exudates can transform into mast cell-like cells under the influence of high levels of specific conditioning factors in their microenvironment. PMID:6824794

  1. Regulatory networks define phenotypic classes of human stem cell lines

    PubMed Central

    Müller, Franz-Josef; Laurent, Louise C.; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Schwartz, Philip H.; Schmidt, Nils O.; Loring, Jeanne F.

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine1, 2 have highlighted the need for a general, reproducible method for classification of these cells3. We report here the creation and analysis of a database of global gene expression profiles (“Stem Cell Matrix”) that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent, and differentiated cell types. Using an unsupervised clustering method4, 5 to categorize a collection of ~150 cell samples, we discovered that pluripotent stem cell lines group together, while other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis6 we uncovered a protein-protein network (“PluriNet”) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas, and induced pluripotent cells). Analysis of published data showed that the PluriNet appears to be a common characteristic of pluripotent cells, including mouse ES and iPS cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotence and self-renewal are under tight control by specific molecular networks. PMID:18724358

  2. Human white blood cells contain cyclobutyl pyrimidine dimer photolyase

    SciTech Connect

    Sutherland, B.M.; Bennett, P.V.

    1995-10-10

    Although enzymatic photoreactivation of cyclobutyl pyrimidine dimers in DNA is present in almost all organisms, its presence in placental mammals is controversial. We tested human white blood cells for photolyase by using three defined DNAs (suprecoiled pET-2, nonsupercoiled bacteriphage {lambda}, and a defined-sequence 287-bp oligonucleotide), two dimer-specific endonucleases (T4 endonuclease V and UV endonuclease from Micrococcus luteus), and three assay methods. We show that human white blood cells contain photolyase that can photorepair pyrimidine dimers in defined supercoiled and linear DNAs and in a 287-bp oligonucleotide and that human photolyase is active on genomic DNA in intact human cells. 44 refs., 3 figs.

  3. Human dental pulp stem cells: Applications in future regenerative medicine

    PubMed Central

    Potdar, Pravin D; Jethmalani, Yogita D

    2015-01-01

    Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells (MSCs) from various human tissues, peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells (DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine. PMID:26131314

  4. Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

    PubMed Central

    Lu, Rong; Bian, Fang; Lin, Jing; Su, Zhitao; Qu, Yangluowa; Pflugfelder, Stephen C.; Li, De-Quan

    2012-01-01

    There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5–14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1×104 in a 35-mm dish (9.6 cm2) grew to confluence (about 1.87–2.41×106 cells) in 12–14 days, representing 187–241 fold expansion with over 7–8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction. PMID:22723892

  5. Asbestos-associated chromosomal changes in human mesothelial cells

    SciTech Connect

    Lechner, J.F.; Tokiwa, T.; LaVeck, M.; Benedict, W.F.; Banks-Schlegel, S.; Yeager, H. Jr.; Banerjee, A.; Harris, C.C.

    1985-06-01

    Replicative cultures of human pleural mesothelial cells were established from noncancerous adult donors. The cells exhibited normal mesothelial cell characteristics including keratin, hyaluronic acid mucin, and long branched microvilli, and they retained the normal human karyotype until senescence. The mesothelial cells were 10 and 100 times more sensitive to the cytotoxic effects of asbestos fibers than normal human bronchial epithelial or fibroblastic cells, respectively. In addition, cultures of mesothelial cells that survived two cytotoxic exposures of amosite fibers were aneuploid with consistent specific chromosomal losses indicative of clonal origin. These aneuploid cells exhibit both altered growth control properties and a population doubling potential of >50 divisions beyond the culture life span (30 doublings) of the control cells.

  6. Resident memory T cells in human health and disease

    PubMed Central

    Clark, Rachael A.

    2015-01-01

    Resident memory T cells are non-recirculating memory T cells that persist long term in epithelial barrier tissues, including the gastrointestinal tract, lung, skin and reproductive tract. Resident memory T cells persist in the absence of antigens, have impressive effector functions and provide rapid on-site immune protection against known pathogens in peripheral tissues. A fundamentally distinct gene expression program differentiates resident memory T cells from circulating T cells. Although these cells likely evolved to provide rapid immune protection against pathogens, autoreactive, aberrantly activated and malignant resident memory cells contribute to numerous human inflammatory diseases including mycosis fungoides and psoriasis. This review will discuss both the science and medicine of resident memory T cells, exploring how these cells contribute to healthy immune function and discussing what is known about how these cells contribute to human inflammatory and autoimmune diseases. PMID:25568072

  7. Derivation of three new human embryonic stem cell lines.

    PubMed

    Bradley, Cara K; Chami, Omar; Peura, Teija T; Bosman, Alexis; Dumevska, Biljana; Schmidt, Uli; Stojanov, Tomas

    2010-04-01

    Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines. PMID:20198447

  8. Purification and cultivation of human pituitary growth hormone secreting cells

    NASA Technical Reports Server (NTRS)

    Hymer, W. C.

    1978-01-01

    The maintainance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro was studied. The primary approach was the testing of agents which may be expected to increase the release of the human growth hormone (hGH). A procedure for tissue procurement is described along with the methodologies used to dissociate human pituitary tissue (obtained either at autopsy or surgery) into single cell suspensions. The validity of the Biogel cell column perfusion system for studying the dynamics of GH release was developed and documented using a rat pituitary cell system.

  9. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  10. Generation of Human Melanocytes from Induced Pluripotent Stem Cells

    PubMed Central

    Okada, Yohei; Akamatsu, Wado; Kuwahara, Reiko; Ohyama, Manabu; Amagai, Masayuki; Matsuzaki, Yumi; Yamanaka, Shinya; Okano, Hideyuki; Kawakami, Yutaka

    2011-01-01

    Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma. PMID:21249204

  11. On the development of extragonadal and gonadal human germ cells

    PubMed Central

    Heeren, A. Marijne; He, Nannan; de Souza, Aline F.; Goercharn-Ramlal, Angelique; van Iperen, Liesbeth; Roost, Matthias S.; Gomes Fernandes, Maria M.; van der Westerlaken, Lucette A. J.; Chuva de Sousa Lopes, Susana M.

    2016-01-01

    ABSTRACT Human germ cells originate in an extragonadal location and have to migrate to colonize the gonadal primordia at around seven weeks of gestation (W7, or five weeks post conception). Many germ cells are lost along the way and should enter apoptosis, but some escape and can give rise to extragonadal germ cell tumors. Due to the common somatic origin of gonads and adrenal cortex, we investigated whether ectopic germ cells were present in the human adrenals. Germ cells expressing DDX4 and/or POU5F1 were present in male and female human adrenals in the first and second trimester. However, in contrast to what has been described in mice, where ‘adrenal’ and ‘ovarian’ germ cells seem to enter meiosis in synchrony, we were unable to observe meiotic entry in human ‘adrenal’ germ cells until W22. By contrast, ‘ovarian’ germ cells at W22 showed a pronounced asynchronous meiotic entry. Interestingly, we observed that immature POU5F1+ germ cells in both first and second trimester ovaries still expressed the neural crest marker TUBB3, reminiscent of their migratory phase. Our findings highlight species-specific differences in early gametogenesis between mice and humans. We report the presence of a population of ectopic germ cells in the human adrenals during development. PMID:26834021

  12. Human satellite cells have regenerative capacity and are genetically manipulable

    PubMed Central

    Marg, Andreas; Escobar, Helena; Gloy, Sina; Kufeld, Markus; Zacher, Joseph; Spuler, Andreas; Birchmeier, Carmen; Izsvák, Zsuzsanna; Spuler, Simone

    2014-01-01

    Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon–mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies. PMID:25157816

  13. Human pluripotent stem cell models of Fragile X syndrome.

    PubMed

    Bhattacharyya, Anita; Zhao, Xinyu

    2016-06-01

    Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism. The causal mutation in FXS is a trinucleotide CGG repeat expansion in the FMR1 gene that leads to human specific epigenetic silencing and loss of Fragile X Mental Retardation Protein (FMRP) expression. Human pluripotent stem cells (PSCs), including human embryonic stem cells (ESCs) and particularly induced PSCs (iPSCs), offer a model system to reveal cellular and molecular events underlying human neuronal development and function in FXS. Human FXS PSCs have been established and have provided insight into the epigenetic silencing of the FMR1 gene as well as aspects of neuronal development. PMID:26640241

  14. [Research with human embryo stem cells. Foundations and judicial limits].

    PubMed

    Eser, Albin; Koch, Hans-Georg

    2004-01-01

    Research with human embryos, and particularly, the use for scientific purposes of human embryonic stem cells has given raise to different sort of problems at the international level. One of the most strict regulation in this field, is this lecture Professors Albin Eser and Hans-Georg Koch analyse the german legal framework in relation with the use of embryos and human embryonic stem cells for scientific purposes. PMID:15544142

  15. Rapid induction of senescence in human cervical carcinoma cells

    NASA Astrophysics Data System (ADS)

    Goodwin, Edward C.; Yang, Eva; Lee, Chan-Jae; Lee, Han-Woong; Dimaio, Daniel; Hwang, Eun-Seong

    2000-09-01

    Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers.

  16. Humanized mice as a model to study human hematopoietic stem cell transplantation.

    PubMed

    Tanner, Anne; Taylor, Stephen E; Decottignies, Wittnee; Berges, Bradford K

    2014-01-01

    Hematopoietic stem cell (HSC) transplantation has the potential to treat a variety of human diseases, including genetic deficiencies, immune disorders, and to restore immunity following cancer treatment. However, there are several obstacles that prevent effective HSC transplantation in humans. These include finding a matched donor, having a sufficient number of cells for the transplant, and the potency of the cells in the transplant. Ethical issues prevent effective research in humans that could provide insight into ways to overcome these obstacles. Highly immunodeficient mice can be transplanted with human HSCs and this process is accompanied by HSC homing to the murine bone marrow. This is followed by stem cell expansion, multilineage hematopoiesis, long-term engraftment, and functional human antibody and cellular immune responses. As such, humanized mice serve as a model for human HSC transplantation. A variety of conditions have been analyzed for their impact on HSC transplantation to produce humanized mice, including the type and source of cells used in the transplant, the number of cells transplanted, the expansion of cells with various protocols, and the route of introduction of cells into the mouse. In this review, we summarize what has been learned about HSC transplantation using humanized mice as a recipient model and we comment on how these models may be useful to future preclinical research to determine more effective ways to expand HSCs and to determine their repopulating potential in vivo. PMID:23962058

  17. The response of human and rodent cells to hyperthermia

    SciTech Connect

    Roizin-Towle, L.; Pirro, J.P. )

    1991-04-01

    Inherent cellular radiosensitivity in vitro has been shown to be a good predictor of human tumor response in vivo. In contrast, the importance of the intrinsic thermosensitivity of normal and neoplastic human cells as a factor in the responsiveness of human tumors to adjuvant hyperthermia has never been analyzed systematically. A comparison of thermal sensitivity and thermo-radiosensitization in four rodent and eight human-derived cell lines was made in vitro. Arrhenius plots indicated that the rodent cells were more sensitive to heat killing than the human, and the break-point was 0.5 degrees C higher for the human than rodent cells. The relationship between thermal sensitivity and the interaction of heat with X rays at low doses was documented by thermal enhancement ratios (TER's). Cells received either a 1 hr exposure to 43 degrees C or a 20 minute treatment at 45 degrees C before exposure to 300 kVp X rays. Thermal enhancement ratios ranged from 1.0 to 2.7 for human cells heated at 43 degrees C and from 2.1 to 5.3 for heat exposures at 45 degrees C. Thermal enhancement ratios for rodent cells were generally 2 to 3 times higher than for human cells, because of the fact that the greater thermosensitivity of rodent cells results in a greater enhancement of radiation damage. Intrinsic thermosensitivity of human cells has relevance to the concept of thermal dose; intrinsic thermo-radiosensitization of a range of different tumor cells is useful in documenting the interactive effects of radiation combined with heat.

  18. STELLA Facilitates Differentiation of Germ Cell and Endodermal Lineages of Human Embryonic Stem Cells

    PubMed Central

    Wongtrakoongate, Patompon; Jones, Mark; Gokhale, Paul J.; Andrews, Peter W.

    2013-01-01

    Stella is a developmentally regulated gene highly expressed in mouse embryonic stem (ES) cells and in primordial germ cells (PGCs). In human, the gene encoding the STELLA homologue lies on chromosome 12p, which is frequently amplified in long-term cultured human ES cells. However, the role played by STELLA in human ES cells has not been reported. In the present study, we show that during retinoic acid (RA)-induced differentiation of human ES cells, expression of STELLA follows that of VASA, a marker of germline differentiation. By contrast, human embryonal carcinoma cells express STELLA at a higher level compared with both karyotypically normal and abnormal human ES cell lines. We found that over-expression of STELLA does not interfere with maintenance of the stem cell state of human ES cells, but following retinoic acid induction it leads to up-regulation of germline- and endodermal-associated genes, whereas neural markers PAX6 and NEUROD1 are down-regulated. Further, STELLA over-expression facilitates the differentiation of human ES cells into BE12-positive cells, in which the expression of germline- and endodermal-associated genes is enriched, and suppresses differentiation of the neural lineage. Taken together, this finding suggests a role for STELLA in facilitating germline and endodermal differentiation of human ES cells. PMID:23457636

  19. Urokinase production by electrophoretically separated cultured human embryonic kidney cells

    NASA Technical Reports Server (NTRS)

    Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

    1985-01-01

    Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

  20. Generating trunk neural crest from human pluripotent stem cells

    PubMed Central

    Huang, Miller; Miller, Matthew L.; McHenry, Lauren K.; Zheng, Tina; Zhen, Qiqi; Ilkhanizadeh, Shirin; Conklin, Bruce R.; Bronner, Marianne E.; Weiss, William A.

    2016-01-01

    Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior “cranial” NCC form craniofacial bone, whereas solely posterior “trunk” NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages. PMID:26812940

  1. Low glucose depletes glycan precursors, reduces site occupancy and galactosylation of a monoclonal antibody in CHO cell culture.

    PubMed

    Villacrés, Carina; Tayi, Venkata S; Lattová, Erika; Perreault, Hélène; Butler, Michael

    2015-07-01

    Controlled feeding of glucose has been employed previously to enhance the productivity of recombinant glycoproteins but there is a concern that low concentrations of glucose could limit the synthesis of precursors of glycosylation. Here we investigate the effect of glucose depletion on the metabolism, productivity and glycosylation of a chimeric human-llama monoclonal antibody secreted by CHO cells. The cells were inoculated into media containing varying concentrations of glucose. Glucose depletion occurred in cultures with an initial glucose ≤5.5 mM and seeded at low density (2.5 × 10(5) cells/mL) or at high cell inoculum (≥2.5 × 10(6) cells/mL) at higher glucose concentration (up to 25 mM). Glucose-depleted cultures produced non-glycosylated Mabs (up to 51%), lower galactosylation index (GI <0.43) and decreased sialylation (by 85%) as measured by mass spectrometry and HPLC. At low glucose a reduced intracellular pool of nucleotides (0.03-0.23 fmoles/cell) was measured as well as a low adenylate energy charge (<0.57). Low glucose also reduced GDP-sugars (by 77%) and UDP-hexosamines (by 90%). The data indicate that under glucose deprivation, low levels of intracellular nucleotides and nucleotide sugars reduced the availability of the immediate precursors of glycosylation. These results are important when applied to the design of fed-batch cultures. PMID:26058832

  2. Somatically Hypermutated Plasmodium-Specific IgM(+) Memory B Cells Are Rapid, Plastic, Early Responders upon Malaria Rechallenge.

    PubMed

    Krishnamurty, Akshay T; Thouvenel, Christopher D; Portugal, Silvia; Keitany, Gladys J; Kim, Karen S; Holder, Anthony; Crompton, Peter D; Rawlings, David J; Pepper, Marion

    2016-08-16

    Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated immunoglobulin M(+) (IgM(+)) and IgG(+) MBC subsets and an unmutated IgD(+) MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM(+) MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge. IgM(+) MBCs also gave rise to T cell-dependent IgM(+) and IgG(+)B220(+)CD138(+) plasmablasts or T cell-independent B220(-)CD138(+) IgM(+) plasma cells. Thus, even in competition with IgG(+) MBCs, IgM(+) MBCs are rapid, plastic, early responders to a secondary Plasmodium rechallenge and should be targeted by vaccine strategies. PMID:27473412

  3. Human gamma delta T-cell recognition of Yersinia enterocolitica.

    PubMed Central

    Young, J L; Goodall, J C; Beacock-Sharp, H; Gaston, J S

    1997-01-01

    We have studied the human gamma delta T-cell response to Yersinia enterocolitica, a facultative intracellular bacterium which causes gastroenteritis and, particularly in human leucocyte antigen (HLA)-B27+ individuals, reactive arthritis (ReA). A marked proliferation of that cytotoxic gamma delta T cells is seen when Yersinia-infected lymphoblastoid cell lines or fixed intact Yersinia are added to cultures of mononuclear cells derived from the synovial fluid of ReA patients or from the peripheral blood of healthy donors. In contrast, heat-inactivated Yersinia fail to stimulate the gamma delta T-cell response. The gamma delta T-cell lines generated killed both autologous and allogeneic infected cell lines. Interestingly, a T-cell line generated from synovial fluid mononuclear cells (SFMC) killed infected autologous cell lines and a cell line matched for HLA-B27 less well than infected allogeneic target cells. gamma delta T-cell clones isolated from this line were found to express V gamma 9V delta 2 T-cell receptor (TCR) and also killed infected mismatched cells more efficiently than autologous targets. Moreover, from experiments using major histocompatability complex (MHC)-deficient cell lines, it was apparent that target cell recognition was MHC independent. Our results suggest that gamma delta T cells can be involved in immunity to Yersinia enterocolitica and should be taken into account when considering immunopathological mechanisms leading to reactive arthritis. PMID:9378487

  4. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  5. On human development: lessons from stem cell systems.

    PubMed

    Medvinsky, Alexander; Livesey, Frederick J

    2015-01-01

    In September 2014, over 100 scientists from around the globe gathered at Wotton House near London for the Company of Biologists' workshop 'From Stem Cells to Human Development'. The workshop covered diverse aspects of human development, from the earliest stages of embryogenesis to differentiation of mature cell types of all three germ layers from pluripotent cells. In this Meeting Review, we summarise some of the exciting data presented at the workshop and draw together the main themes that emerged. PMID:25516966

  6. Neoplastic transformation of human diploid fibroblast cells by chemical carcinogens

    PubMed Central

    Kakunaga, Takeo

    1978-01-01

    Cultured fibroblast cells derived from a skin biopsy sample taken from normal human adult were exposed to a potent carcinogen, 4-nitroquinoline 1-oxide. Alterations of cell growth pattern such as higher density and piling up of cells were noticed in some fractions of cultures that were successively subcultured after nitroquinoline oxide treatment. Morphologically altered cells retained this growth pattern and became established lines of transformed cells without showing the limited life-span characteristic of normal cells in culture. The transformed cells showed a higher saturation density and the ability to grow in soft agar, properties that are usually correlated with neoplastic transformation of cells in culture. Selection of preexisting transformed human cells as a mechanism of this observed transformation seemed unlikely because clones of these normal cells could also be used to assess the transforming effect of nitroquinoline oxide. Preliminary results suggest that numerous cell divisions were required for the development of the transformation after nitroquinoline oxide treatment of these human cells. When the transformed cell lines were injected subcutaneously into nude (athymic) mice, solid tumors were produced at the site of inoculation. Treatment with N-methyl-N′-nitro-N-nitrosoguanidine also induced cell transformation, in a manner similar to treatment with nitroquinoline oxide. However, transformation was not induced with (i) 4-aminoquinoline 1-oxide (a noncarcinogenic derivative of 4-nitroquinoline 1-oxide), (ii) 3-methylcholanthrene (a carcinogen that cannot be metabolically activated by the target cells employed), or (iii) the solvent dimethyl sulfoxide. Images PMID:418410

  7. 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. PMID:26246271

  8. Piperlongumine Suppresses Proliferation of Human Oral Squamous Cell Carcinoma through Cell Cycle Arrest, Apoptosis and Senescence.

    PubMed

    Chen, San-Yuan; Liu, Geng-Hung; Chao, Wen-Ying; Shi, Chung-Sheng; Lin, Ching-Yen; Lim, Yun-Ping; Lu, Chieh-Hsiang; Lai, Peng-Yeh; Chen, Hau-Ren; Lee, Ying-Ray

    2016-01-01

    Oral squamous cell carcinoma (OSCC), an aggressive cancer originating in the oral cavity, is one of the leading causes of cancer deaths in males worldwide. This study investigated the antitumor activity and mechanisms of piperlongumine (PL), a natural compound isolated from Piper longum L., in human OSCC cells. The effects of PL on cell proliferation, the cell cycle, apoptosis, senescence and reactive oxygen species (ROS) levels in human OSCC cells were investigated. PL effectively inhibited cell growth, caused cell cycle arrest and induced apoptosis and senescence in OSCC cells. Moreover, PL-mediated anti-human OSCC behavior was inhibited by an ROS scavenger N-acetyl-l-cysteine (NAC) treatment, suggesting that regulation of ROS was involved in the mechanism of the anticancer activity of PL. These findings suggest that PL suppresses tumor growth by regulating the cell cycle and inducing apoptosis and senescence and is a potential chemotherapy agent for human OSCC cells. PMID:27120594

  9. Piperlongumine Suppresses Proliferation of Human Oral Squamous Cell Carcinoma through Cell Cycle Arrest, Apoptosis and Senescence

    PubMed Central

    Chen, San-Yuan; Liu, Geng-Hung; Chao, Wen-Ying; Shi, Chung-Sheng; Lin, Ching-Yen; Lim, Yun-Ping; Lu, Chieh-Hsiang; Lai, Peng-Yeh; Chen, Hau-Ren; Lee, Ying-Ray

    2016-01-01

    Oral squamous cell carcinoma (OSCC), an aggressive cancer originating in the oral cavity, is one of the leading causes of cancer deaths in males worldwide. This study investigated the antitumor activity and mechanisms of piperlongumine (PL), a natural compound isolated from Piper longum L., in human OSCC cells. The effects of PL on cell proliferation, the cell cycle, apoptosis, senescence and reactive oxygen species (ROS) levels in human OSCC cells were investigated. PL effectively inhibited cell growth, caused cell cycle arrest and induced apoptosis and senescence in OSCC cells. Moreover, PL-mediated anti-human OSCC behavior was inhibited by an ROS scavenger N-acetyl-l-cysteine (NAC) treatment, suggesting that regulation of ROS was involved in the mechanism of the anticancer activity of PL. These findings suggest that PL suppresses tumor growth by regulating the cell cycle and inducing apoptosis and senescence and is a potential chemotherapy agent for human OSCC cells. PMID:27120594

  10. 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