208 CYTOLYTIC ANALYSIS AND NUCLEAR TRANSFER OF hCD46-TRANSGENIC PORCINE EMBRYONIC GERM CELLS TO DEVELOP AND IN VITRO MODEL OF XENOTRANSPLANTATION

2006 ◽  
Vol 18 (2) ◽  
pp. 212
Author(s):  
J. Y. Won ◽  
K. S. Ahn ◽  
S. Y. Heo ◽  
J. H. Kang ◽  
H. Shim
Keyword(s):  
Human Serum ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
In Vitro Model ◽  
Regulatory Protein ◽  
Cell Types ◽  
Human Complement ◽  
Transgenic Pigs ◽  
Vitro Model

Pigs are considered the most likely source of organs for xenotransplantation due to their anatomical and physiological similarities to humans. Production of transgenic pigs including addition of human complement-regulatory protein genes and deletion of alpha-1,3-galactosyl transferase gene may overcome hyperacute rejection (HAR), the first and currently the most critical immunological hurdle in the development of xenogeneic organs for human transplantation. However, even after resolving HAR in pig-to-human xenotransplantation, a series of other transgenic pigs may be required to alleviate subsequent acute and chronic rejection and incompatibility of porcine proteins to human counterparts. The production of transgenic pigs is not only labor-intensive, time-consuming, and costly, but also the usefulness of such pigs in transplantation to humans is unpredictable. For these reasons, development of a reliable in vitro procedure to pre-evaluate effectiveness of the transgenic approach would be beneficial. This study was preformed to establish an in vitro model of xenotransplantation using porcine embryonic germ (EG) cells, undifferentiated stem cells derived from culture of primordial germ cells. Porcine EG cells were maintained in feeder-free state in DMEM containing 15% (v/v) fetal bovine serum and 1000 units/mL leukemia inhibitory factor. Human complement down-regulator hCD46 (also known as MCP, membrane cofactor protein) gene under the regulation of cytomegalovirus promoter was introduced into porcine EG cells. Transfected cells were selected by antibiotic treatment and confirmed by PCR. To test the resistance of hCD46-transgenic EG cells to human xenoreactive natural antibody and complement, EG cells were cultured for 1.5 days in DMEM containing 15% (v/v) normal human serum. The treatment with human serum did not affect the survival of hCD46-transgenic EG cells, whereas with the same treatment approximately one half of non-transfected EG cells failed to survive (P < 0.01). Transgenic EG cells presumably capable of overcoming HAR were used as nuclear donors for subsequent transfer of nuclei into enucleated oocytes. Among 110 reconstituted oocytes, 19 (17.3%) developed to the blastocyst stage. Analysis of individual nuclear transfer embryos by PCR indicated that 89.5% (17/19) of embryos contained transgene hCD46. The PCR-negative embryos might be due to an incomplete antibiotic selection of cells after transfection. Overall, the results of the present study demonstrate that the cell culture-based model of xenotransplantation may validate the usefulness of particular transgenic pigs prior to actual production. Further experiments on differentiation of transgenic EG cells into various cell types, cytolytic analysis of such cells to assess efficiency of xenotransplantation, and subsequent production and transfer of transgenic clone embryos to recipients may provide a useful new procedure to accelerate xenotransplantation research.

scholarly journals Rho family GTPase Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells

2003 ◽  
Vol 372 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Nathalie NAUD ◽  
Aminata TOURÉ ◽  
Jianfeng LIU ◽  
Charles PINEAU ◽  
Laurence MORIN ◽  
...  
Keyword(s):  
Germ Cells ◽  
In Vitro Model ◽  
Cell Types ◽  
Glutathione S Transferase ◽  
Rac Gtpase ◽  
Male Germ Cells ◽  
Rho Family ◽  
Vitro Model ◽  
Null Mutant Mice

The male-germ-cell Rac GTPase-activating protein gene (MgcRacGAP) was initially described as a human RhoGAP gene highly expressed in male germ cells at spermatocyte stage, but exhibits significant levels of expression in most cell types. In somatic cells, MgcRacGAP protein was found to both concentrate in the midzone/midbody and be required for cytokinesis. As a RhoGAP, MgcRacGAP has been proposed to down-regulate RhoA, which is localized to the cleavage furrow and midbody during cytokinesis. Due to embryonic lethality in MgcRacGAP-null mutant mice and to the lack of an in vitro model of spermatogenesis, nothing is known regarding the role and mode of action of MgcRacGAP in male germ cells. We have analysed the expression, subcellular localization and molecular interactions of MgcRacGAP in male germ cells. Whereas MgcRacGAP was found only in spermatocytes and early spermatids, the widespread RhoGTPases RhoA, Rac1 and Cdc42 (which are, to various extents, in vitro substrates for MgcRacGAP activity) were, surprisingly, not detected at these stages. In contrast, Rnd2, a Rho family GTPase-deficient G-protein was found to be co-expressed with MgcRacGAP in spermatocytes and spermatids. MgcRacGAP was detected in the midzone of meiotic cells, but also, unexpectedly, in the Golgi-derived pro-acrosomal vesicle, co-localizing with Rnd2. In addition, a stable Rnd2–MgcRacGAP molecular complex could be evidenced by glutathione S-transferase pull-down and co-immunoprecipitation experiments. We conclude that Rnd2 is a probable physiological partner of MgcRacGAP in male germ cells and we propose that MgcRacGAP, and, quite possibly, other RhoGAPs, may participate in signalling pathways involving Rnd family proteins.

scholarly journals A Central Role for the Armadillo Protein Plakoglobin in the Autoimmune Disease Pemphigus Vulgaris

2001 ◽  
Vol 153 (4) ◽  
pp. 823-834 ◽  
Author(s):  
Reto Caldelari ◽  
Alain de Bruin ◽  
Dominique Baumann ◽  
Maja M. Suter ◽  
Christiane Bierkamp ◽  
...  
Keyword(s):  
Steric Hindrance ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Pemphigus Vulgaris ◽  
Cell Types ◽  
Linear Distribution ◽  
Igg Binding ◽  
Vitro Model ◽  
First Time

In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG−/−) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG−/− cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG−/− keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.

scholarly journals CELLULAR IMMUNITY IN VITRO

1971 ◽  
Vol 133 (6) ◽  
pp. 1377-1389 ◽  
Author(s):  
Harvey B. Simon ◽  
John N. Sheagren
Keyword(s):  
Cellular Immunity ◽  
In Vitro Model ◽  
Specific Antigen ◽  
Cell Types ◽  
Intracellular Bacteria ◽  
Migration Inhibition ◽  
Vitro Model ◽  
And Control ◽  
Macrophage Migration Inhibition

An in vitro model of cellular immunity in the guinea pig was established. Animals were immunized with tubercle bacilli, bovine gamma globulin, or picrylated human serum albumin in complete Freund's adjuvant. Oil-induced peritoneal exudates from immune and control animals were cultured overnight with and without specific antigen. The cultures were washed and the macrophage monolayers were infected with Listeria monocytogenes. At intervals the monolayers were lysed and the numbers of viable intracellular bacteria were quantitated by pour plate cultures. Random monolayers were also evaluated in sequence by visually counting the intracellular bacteria on Gram-stained plates. Both methods demonstrated that the macrophages from immune animals had markedly enhanced listericidal activity when the peritoneal exudates were cultured with antigen before infection. Macrophage migration inhibition was also demonstrated under these conditions. The experiments reported here describe an in vitro model of cellular immunity which will allow separation and recombination of cell types and direct assay of cell products in efforts to elucidate further the mechanisms of the immunologically mediated enhancement of macrophage bactericidal capacity.

TT30, a Novel Human Complement Inhibitor in Development for Paroxysmal Nocturnal Hemoglobinuria and Other Hemolytic Disorders, Demonstrates Red Blood Cell Surface Targeting and Retention In a Model of Complement Alternative Pathway-Mediated Hemolysis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 638-638
Author(s):  
Masha Fridkis-Hareli ◽  
Michael Storek ◽  
Antonio M. Risitano ◽  
Ante S. Lundberg ◽  
Christopher J Horvath ◽  
...  
Keyword(s):  
Cell Surface ◽  
Human Serum ◽  
Alternative Pathway ◽  
Human Complement ◽  
Advisory Committees ◽  
Complement Alternative Pathway ◽  
Flow Cytometric ◽  
Vitro Model ◽  
The Complement System

Abstract Abstract 638 Polymorphisms and mutations that promote Complement Alternative Pathway (CAP) activity are associated with human diseases, especially genetically linked hemolytic disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and thrombotic microangiopathy (TMA) disorders such as atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura. The complement system can be activated through three unique pathways (classical, lectin/mannose and alternative). In PNH, the lack of CD55 on RBC allows CAP-initiated complement C3 activation by C3 convertases, while the lack of CD59 allows C5 activation by C5 convertase to proceed to formation of the membrane attack complex (MAC; C5b-9), resulting in intravascular hemolysis (IVH). Treatment of patients with the anti-C5 monoclonal antibody (mAb) eculizumab abrogates IVH; however, because eculizumab does not inhibit CAP activity prior to C5, covalently bound C3 fragments (C3frag) and both C3 and C5 convertases continue to accumulate on PNH red blood cells (RBCs). Clearance of PNH RBCs that are C3frag-coated by complement receptors within the reticuloendothelial system (RES) is the putative cause of continued extravascular hemolysis (EVH) in patients who receive eculizumab. Continued anemia and transfusion requirements are found in a substantial proportion of eculizumab-treated patients, and correlate with PNH RBC-bound C3frag. High levels of C5 convertases on the same cells may also contribute to intermittent escape from eculizumab control of IVH due to pharmacodynamic breakthrough. To selectively modulate CAP activity on PNH RBC and replace the CD55-mediated control of CAP activation, we developed TT30, a novel therapeutic fusion protein linking the C3frag-binding domain of human complement receptor type 2 (CR2/CD21) with the CAP inhibitory domain of human factor H (fH). TT30 delivers cell surface-targeted (via CR2) inhibition of CAP activity (via fH) and blocks the ex vivo hemolysis of PNH RBCs, while at the same time retaining the normal ability of the complement system to efficiently activate C3 through the classical and lectin pathways. We studied the mechanism of TT30 prevention of hemolysis by control of CAP activity in human serum using: 1) an in vitro model of CAP-mediated hemolysis in which rabbit RBCs are exposed to normal human serum under conditions promoting CAP activation (Mg++/EGTA) and the extent of hemolysis is quantified by measuring hemoglobin release; 2) flow cytometric phenotyping of C3frag accumulation on rabbit RBCs exposed to normal or C5-deficient human serum using mAbs specific for human iC3b (A710, Quidel) or C3d (A702, Quidel); 3) flow cytometric demonstration of TT30 binding to C3frag+ rabbit RBCs with a noncompeting mAbs against CR2 (HB5, Taligen) or fH (A255, Quidel); and 4) an in vitro model of CAP-mediated MAC formation in which human serum is exposed to an LPS-coated surface in the presence of Mg++/EGTA and CAP activation through to the MAC is quantified by detection of a neoantigen in poly-C9 by ELISA. The results demonstrate that TT30 efficiently inhibits CAP-mediated MAC formation (IC50 of 3.2 ug/ml) and hemolysis (IC50 of 50.1 ug/ml) and that both of these activities are dependent upon targeting to C3frag+ surfaces by CR2, as evidenced by complete reversal of TT30 inhibitory activity in the presence of a 2-fold molar excess of a competing anti-CR2 mAb (1048, Taligen). Rabbit RBCs were shown to become coated with C3frag in the presence of normal and C5-deficient serum and to undergo lysis with normal serum. TT30 was readily demonstrated to be bound to C3frag+ RBCs during prevention of hemolysis and to remain detectable on RBCs for at least 24 hours. The amount of bound TT30 was proportional to the accumulation of C3frags. Collectively, these results demonstrate that TT30 displays targeted control of cell surface CAP activation, with both effective and prolonged blockade of MAC formation, and dose-dependent inhibition of hemolysis. Therefore, the CAP-specific novel therapeutic TT30 has potential utility for the treatment of human complement-mediated diseases, such as PNH and aHUS, in which modulation of CAP activation is predicted to be clinically beneficial. Disclosures: Fridkis-Hareli: Taligen Therapeutics: Employment. Storek:Taligen Therapeutics: Employment. Risitano:Taligen Therapeutics: Consultancy, Research Funding. Lundberg:Taligen Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees. Horvath:Taligen Therapeutics: Employment. Holers:Taligen Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

scholarly journals Efficient differentiation of human primordial germ cells through geometric control reveals a key role for NODAL signaling

2021 ◽  
Author(s):  
Kyoung Jo ◽  
Seth Teague ◽  
Bohan Chen ◽  
Hina Aftab Khan ◽  
Emily Freeburne ◽  
...  
Keyword(s):  
Germ Cells ◽  
In Vitro Model ◽  
Primordial Germ Cells ◽  
Bmp Signaling ◽  
Critical Parameters ◽  
Nodal Signaling ◽  
Relative Timing ◽  
Vitro Model ◽  
First Time

Human primordial germ cells (hPGCs) form around the time of implantation and are the precursors of eggs and sperm. Many aspects of hPGC specification remain poorly understood. Here we show that micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 give rise to hPGC-like cells (hPGCLC) and use these as a quantitatively reproducible and simple in vitro model to interrogate this important developmental event. We characterize micropatterned hPSCs up to 96h for the first time and show that hPGCLC populations are stable and continue to mature. By perturbing signaling during hPGCLC differentiation, we identify a previously unappreciated role for NODAL signaling and find that the relative timing and duration of BMP and NODAL signaling are critical parameters controlling the number of hPGCLCs. We formulate a mathematical model for a network of cross-repressive fates driven by NODAL and BMP signaling which predicts the measured fate patterns after signaling perturbations. Finally, we show that hPSC colony size dictates the efficiency of hPGCLC specification, which led us to dramatically improve the efficiency of hPGCLC differentiation over current protocols.

In vitro development of nuclear transfer embryos derived from porcine embryonic germ cells and their descendent neural precursor cells

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Susa Shin ◽  
Kwang Sung Ahn ◽  
Seong-Jun Choi ◽  
Soon Young Heo ◽  
Hosup Shim
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Somatic Cells ◽  
Cell Types ◽  
Neural Precursor ◽  
Blastocyst Development ◽  
Donor Cells

SummaryUndifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.

scholarly journals Pancreatic Precursors and Differentiated Islet Cell Types From Murine Embryonic Stem Cells: An In Vitro Model to Study Islet Differentiation

Diabetes ◽  
2003 ◽  
Vol 52 (8) ◽  
pp. 2016-2024 ◽  
Author(s):  
B. W. Kahan ◽  
L. M. Jacobson ◽  
D. A. Hullett ◽  
J. M. Ochoada ◽  
T. D. Oberley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Islet Cell ◽  
In Vitro Model ◽  
Embryonic Stem ◽  
Cell Types ◽  
Murine Embryonic Stem Cells ◽  
Vitro Model ◽  
Islet Cell Types

Insulin resistance in uremia: In vitro model in the rat liver using human serum to study mechanisms

Metabolism ◽  
1986 ◽  
Vol 35 (11) ◽  
pp. 989-998 ◽  
Author(s):  
Franco Folli ◽  
Madhur K. Sinha ◽  
Diego Brancaccio ◽  
Jose F. Caro
Keyword(s):  
Insulin Resistance ◽  
Human Serum ◽  
Rat Liver ◽  
In Vitro Model ◽  
Vitro Model
Sign in / Sign up

Export Citation Format

Share Document