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

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.

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Examining the Developmental Trajectory of an in Vitro Model of Mouse Primordial Germ Cells following Exposure to Environmentally Relevant Bisphenol A Levels

Environmental Health Perspectives ◽

10.1289/ehp8196 ◽

2021 ◽

Vol 129 (9) ◽

Author(s):

Steen K.T. Ooi ◽

Hui Jiang ◽

Yanyuan Kang ◽

Patrick Allard

Keyword(s):

Bisphenol A ◽

Germ Cells ◽

In Vitro Model ◽

Primordial Germ Cells ◽

Developmental Trajectory ◽

Vitro Model

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

The Journal of Cell Biology ◽

10.1083/jcb.153.4.823 ◽

2001 ◽

Vol 153 (4) ◽

pp. 823-834 ◽

Cited By ~ 130

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.

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SPINK1-induced amelioration of impaired autophagy contributes to suppression of trypsinogen activation in a model of acute pancreatitis

10.1101/2020.03.06.981654 ◽

2020 ◽

Author(s):

Yuxiao Zhao ◽

Jianlong Jia ◽

Abdullah Shopit ◽

Yang Liu ◽

Jun Wang

Keyword(s):

Acute Pancreatitis ◽

In Vitro Model ◽

Autophagic Flux ◽

Trypsin Activity ◽

Autophagy Flux ◽

Trypsinogen Activation ◽

Vitro Model ◽

First Time ◽

Induced Amelioration

AbstractSPINK1 has been regarded as a reversible trypsinogen inhibitor for the inappropriate activation of trypsin, a key step in the initiation of acute pancreatitis (AP). However, the mechanisms of its action remains largely unclear and controversial. Here, we reported an unexpected effects of SPINK1 on inhibiting trypsinogen activation through the regulation of impaired autophagy in cerulein-stimulated AR42J cells, a well-established in vitro model of acute pancreatitis. Firstly, we found that the impaired autophagic flux was induced and trypsinogen activity enhanced in the above setting. Then, we showed that SPINK1 overexpression could inhibit the level of increased autophagic activity, improving the hindered autophagy flux, and significantly decreased the trypsinogen activity, whereas shRNA-caused downregulation of SPINK1 exacerbated the impairment of autophagic flux and trypsin activity, in the same cerulein-processed cells. More importantly, the trypsinogen activation in this model could be ameliorated by 3-Methyladenine(3-MA), an autophagy inhibitor. Thus, this study revealed, possibly for the first time, that SPINK1 greatly blocked the trypsinogen activation possibly through the modulation of impaired autophagy in cerulein-induced in vitro model of acute pancreatitis.

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Iron-related transcriptomic variations in CaCo-2 cells, an in vitro model of intestinal absorptive cells

Physiological Genomics ◽

10.1152/physiolgenomics.00297.2005 ◽

2006 ◽

Vol 26 (1) ◽

pp. 55-67 ◽

Cited By ~ 7

Author(s):

Céline Chicault ◽

Bertrand Toutain ◽

Annabelle Monnier ◽

Marc Aubry ◽

Patricia Fergelot ◽

...

Keyword(s):

Gene Expression ◽

Functional Annotation ◽

In Vitro Model ◽

Iron Absorption ◽

Intracellular Iron ◽

Absorptive Cells ◽

Number Of Genes ◽

Vitro Model ◽

First Time

Regulation of iron absorption by duodenal enterocytes is essential for the maintenance of homeostasis by preventing iron deficiency or overload. Despite the identification of a number of genes implicated in iron absorption and its regulation, it is likely that further factors remain to be identified. For that purpose, we used a global transcriptomic approach, using the CaCo-2 cell line as an in vitro model of intestinal absorptive cells. Pangenomic screening for variations in gene expression correlating with intracellular iron content allowed us to identify 171 genes. One hundred nine of these genes are clustered into five types of expression profile. This is the first time that most of these genes have been associated with iron metabolism. Functional annotation of these five clusters indicates potential links between the immune response, proteolysis processes, and iron depletion. In contrast, iron overload is associated with cellular metabolism, especially that of lipids and glutathione involving redox function and electron transfer.

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

Biochemical Journal ◽

10.1042/bj20021652 ◽

2003 ◽

Vol 372 (1) ◽

pp. 105-112 ◽

Cited By ~ 27

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.

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Pluripotent stem cells of the mouse as a potential in vitro model for mammalian germ cells. Sister chromatid exchanges induced by MMC and ENU in undifferentiated cell lines compared to differentiated cell lines

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/s1383-5718(99)00090-x ◽

1999 ◽

Vol 444 (1) ◽

pp. 97-102 ◽

Cited By ~ 6

Author(s):

Susanne Bremer ◽

Richard Vogel

Keyword(s):

Stem Cells ◽

Cell Lines ◽

Germ Cells ◽

Pluripotent Stem Cells ◽

In Vitro Model ◽

Sister Chromatid Exchanges ◽

Undifferentiated Cell ◽

Vitro Model ◽

Chromatid Exchanges

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

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab208 ◽

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.

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