WNT3A signalling pathway in buffalo (Bubalus bubalis) embryonic stem cells

2014 ◽  
Vol 26 (4) ◽  
pp. 551 ◽  
Author(s):  
Mohammad Zandi ◽  
Musharifa Muzaffar ◽  
Syed Mohmad Shah ◽  
Ramakant Kaushik ◽  
Manoj Kumar Singh ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Polymerase Chain Reaction Analysis ◽  
Bubalus Bubalis ◽  
Embryoid Bodies ◽  
Signalling Pathway ◽  
Leukaemia Inhibitory Factor ◽  
In Vitro Fertilisation ◽  
Canonical Wnt

The aim of this study was to investigate the transcriptional profile and role of WNT3A signalling in maintaining buffalo embryonic stem (ES) cells in a pluripotent state and in the induction of their differentiation. ES cells were derived from embryos produced by in vitro fertilisation (iESC), parthenogenesis (pESC) and hand-made cloning (cESC). The expression of WNT3A, its receptors and intermediate signalling pathways were found to be conserved in ES cells derived from the three different sources. WNT3A was expressed in ES cells but not in embryoid bodies derived from iESC or in buffalo fetal fibroblast cells. It was revealed by real-time polymerase chain reaction analysis that following supplementation of culture medium with WNT3A (100, 200 or 400 ng mL–1) a significant increase (P < 0.05) was observed in the expression level of β-CATENIN, which indicated the activation of the canonical WNT pathway. WNT3A, in combination with exogenous fibroblast growth factor-2 and leukaemia inhibitory factor, induced proliferation of undifferentiated ES cells. Differentiation studies showed that WNT3A caused formation of scaffold-like structures and inhibition of differentiation into neuron-like cells. In conclusion, the WNT3A signalling pathway is necessary both for maintaining undifferentiated buffalo ES cells as well as for directing their differentiation.

Enrichment of blood from embryonic stem cells in vitro

1998 ◽  
Vol 10 (8) ◽  
pp. 563 ◽  
Author(s):  
Andrew C. Perkins
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Embryonic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Leukaemia Inhibitory Factor ◽  
Chimeric Mouse ◽  
Differentiation Potential

Murine embryonic stem (ES) cells are pluripotent. When injected into blastocysts they can give rise to every cell type of a derived chimeric mouse including germ cells. Embryonic stem cells also possess remarkable in vitro differentiation potential. When removed from stromal support and leukaemia inhibitory factor (LIF), ES cells differentiate into structures known as embryoid bodies (EBs), in which all three germ layers develop and interact. As ES cells from humans become available there is increasing interest in the potential for EBs to provide an unlimited supply of stem cells for somatic transplantation therapies. Realisation of this potential will require greater understanding of the molecular determinants of cell fate within EBs. Also, culture techniques for selective expansion of cell lineages of interest will reduce the risks associated with transplantation of EB-derived cells. In this paper the kinetics of expression of mRNA and protein for early mesoderm markers within EBs is reported. In addition, a three-step culture system incorporating co-cultivation on the bone marrow derived stromal cell line, MC3T3-G2/PA6 (PA6), is explored as a way to select for haematopoietic progenitor cells (HPCs) and against undifferentiated ES cells. A system like this could enhance purification of haematopoietic stem cells (HSCs) from ES cells for bone marrow transplantation.

Abstract 1457: IGFBP-4-Mediated Wnt Inhibition is Required for Cardiac Myogenesis

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Weidong Zhu ◽  
Ichiro Shiojima ◽  
Li Zhi ◽  
Hiroyuki Ikeda ◽  
Masashi Yoshida ◽  
...  
Keyword(s):  
Growth Factor ◽  
Wnt Signaling ◽  
Heart Diseases ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cardiomyocyte Differentiation ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt

Insulin-like growth factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs). Although some of the effects of IGFBPs appear to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown. In this study we demonstrate that IGFBP-4 is a novel cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation of P19CL6 embryonal carcinoma cells and embryonic stem (ES) cells in vitro. Conversely, siRNA-mediated knockdown of IGFBP-4 in P19CL6 cells or ES cells attenuated cardiomyocyte differentiation, and morpholino-mediated knockdown of IGFBP-4 in Xenopus embryos resulted in severe cardiac defects and complete absence of the heart in extreme cases. We also demonstrate that the cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signaling. IGFBP-4 physically interacted with a Wnt receptor Frizzled 8 (Frz8) and a Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Moreover, the cardiogenic defects induced by IGFBP-4 knockdown both in vitro and in vivo was rescued by simultaneous inhibition of canonical Wnt signaling. Thus, IGFBP-4 is an inhibitor of the canonical Wnt signaling, and Wnt inhibition by IGFBP-4 is required for cardiogenesis. The present study provides a molecular link between IGF signaling and Wnt signaling, and suggests that IGFBP-4 may be a novel therapeutic target for heart diseases.

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2740-2749 ◽  
Author(s):  
CD Helgason ◽  
G Sauvageau ◽  
HJ Lawrence ◽  
C Largman ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Embryoid Bodies ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

scholarly journals Isolation, Characterization and Differentiation Potential of Chicken Spermatogonial Stem Cell Derived Embryoid Bodies

2016 ◽  
Vol 16 (1) ◽  
pp. 115-128 ◽  
Author(s):  
Thanh Luan Nguyen ◽  
Jae Gyu Yoo ◽  
Neelesh Sharma ◽  
Sung Woo Kim ◽  
Yong Jun Kang ◽  
...  
Keyword(s):  
Developmental Biology ◽  
Regenerative Medicine ◽  
Connexin 43 ◽  
Es Cells ◽  
Periodic Acid ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Significant Finding ◽  
Differentiation Potential

Abstract Human, murine and monkey spermatogonial stem cells (SSCs) have the capability to undergo self-renewal and differentiation into different body cell types in vitro, which are expected to serve as a powerful tool and resource for the developmental biology and regenerative medicine. We have successfully isolated and characterized the chicken SSCs from 3-day-old chicken testicular cells. The pluripotency was using Periodic Acid-Schiff (PAS ) staining or alkaline phosphatase staining, and antibodies to stage-specific embryonic antigens. In suspension culture conditions SSCs formed embryoid bodies (EBs) like embryonic stem (ES) cells. Subsequently EB differentiated into osteoblasts, adipocytes and most importantly into cardiomyocytes under induced differentiation conditions. The differentiation potential of EBs into cardiomyocyte-like cells was confirmed by using antibodies against sarcomeric α-actinin, cardiac troponin T and connexin 43. Cardiomyocytes-like cells were also confirmed by RT-PCR analysis for several cardiac cell genes like GATA-4, Nkx2-5, α-MHC, and ANF. We have successfully established an in vitro differentiation system for chicken SSCs into different body cells such as osteoblasts, adipocytes and cardiomyocytes. The most significant finding of this study is the differentiation potential of chicken SSCs into cardiomyocytes. Our findings may have implication in developmental biology and regenerative medicine by using chicken as the most potential animal model.

Targeted Disruption of the Mouse Mitoferrin (Slc25A37) Mitochondrial Solute Carrier Results in Defective Primitive and Definitive Erythropoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 265-265 ◽  
Author(s):  
Barry H. Paw ◽  
Babette Gwynn ◽  
Nathaniel B. Langer ◽  
George C. Shaw ◽  
Amy J. Lambert ◽  
...  
Keyword(s):  
Aminolevulinic Acid ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Embryonic Lethality ◽  
In Vitro Differentiation ◽  
Mitochondrial Iron ◽  
Solute Carrier ◽  
Definitive Erythropoiesis

Abstract We previously described a zebrafish mutant, frascati (frs), which exhibits profound hypochromic anemia and erythroid maturation arrest due to defects in mitochondrial iron uptake. Through positional cloning, we showed that the frs gene encodes a novel member of the vertebrate mitochondrial solute carrier family (SLC25), mitoferrin (mfrn, slc25a37). Mfrn, which is highly expressed in fetal and adult hematopoietic tissues of zebrafish and mouse, functions as the major mitochondrial iron importer essential for heme biosynthesis in vertebrate erythroblasts (Shaw GC, et al. 2006 Nature 440:96–100). To study the function of Mfrn in mammalian organisms, we identified an embryonic stem (ES) cell clone that harbors a gene trap b-geo cassette in intron 1 that inactivates the Mfrn locus. Homozygous disruption of the Mfrn locus results in embryonic lethality at E11.5 from profound anemia due to a failure of primitive erythropoiesis, confirming the requirement of Mfrn in mammalian development . Circumventing the embryonic lethality, we generated Mfrn−/− ES cells to study the role of Mfrn in definitive erythropoiesis by in vitro differentiation of embryoid bodies and mixed chimera assays. Mfrn−/− ES cells were defective in promoting the growth, differentiation, and hemoglobinization of both primitive and definitive erythroblasts by in vitro differentiation of embryoid bodies. In mixed chimera studies, Mfrn−/− ES cells failed to contribute to the erythroid compartment of adult mosaic mice, whereas measurable contribution of Mfrn−/− donor cells could be assayed in the non-erythroid, leukocyte compartment. Transcriptome microarray analysis, using the mouse Affymetrix GeneChip and the custom IronChip, revealed unexpected down-regulation of transcripts for heme-biosynthetic enzymes in Mfrn−/− erythroblasts. The block in protoprophyrin synthesis, as well as mitochondrial heme synthesis, could be partially rescued by the addition of aminolevulinic acid (ALA) to Mfrn−/− erythroblasts in vitro. Our data demonstrate that mitochondrial iron homeostasis, working through the Mfrn iron importer, coordinately regulates the synthetic pathways for porphyrin and heme in developing mammalian erythroblasts.

DNA synthesis and multinucleation in embryonic stem cell-derived cardiomyocytes

1995 ◽  
Vol 269 (6) ◽  
pp. H1913-H1921 ◽  
Author(s):  
M. G. Klug ◽  
M. H. Soonpaa ◽  
L. J. Field
Keyword(s):  
Dna Synthesis ◽  
Thymidine Incorporation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Isolated Cells ◽  
In Vitro System ◽  
Multinucleated Cells

The proliferative capacity of embryonic stem (ES) cell-derived cardiomyocytes was assessed. Enriched preparations of cardiomyocytes were isolated by microdissection of the cardiogenic regions of cultured embryoid bodies. The identity of the isolated cells was established by immunocytology, and mitotic activity was monitored by [3H]thymidine incorporation and pulse-chase experiments. ES-derived cardiomyocytes were mitotically active and predominantly mononucleated at 11 days after cardiogenic induction. By 21 days postinduction, cardiomyocyte DNA synthesis was markedly decreased, with a concomitant increase in the percentage of multinucleated cells. Interestingly, the duration of active cardiomyocyte reduplication in the ES system appeared to be roughly similar to that observed during normal murine cardiogenesis. Given these observations, as well as the genetic tractability of ES cells, ES-derived cardiogenesis might provide a useful in vitro system with which to assess the molecular regulation of the cardiomyocyte cell cycle.

Role of the phosphoinositide 3-kinase pathway in mouse embryonic stem (ES) cells

2005 ◽  
Vol 33 (6) ◽  
pp. 1522-1525 ◽  
Author(s):  
K. Takahashi ◽  
M. Murakami ◽  
S. Yamanaka
Keyword(s):  
Es Cells ◽  
Mammalian Target Of Rapamycin ◽  
Embryonic Stem ◽  
Pi3k Pathway ◽  
Leukaemia Inhibitory Factor ◽  
Mouse Es Cells ◽  
Ras Family ◽  
Phosphoinositide 3 Kinase ◽  
Kinase Pathway

Mouse ES (embryonic stem) cells maintain pluripotency with robust proliferation in vitro. ES cells share some similarities with cancer cells, such as anchorage-independent growth, loss of contact inhibition and tumour formation. After differentiation, ES cells lose pluripotency and tumorigenicity. Recent studies showed that the PI3K (phosphoinositide 3-kinase) pathway is important for proliferation, survival and maintenance of pluripotency in ES cells. The PI3K pathway is activated by growth factors and cytokines including insulin and leukaemia inhibitory factor. In addition to these exogenous factors, the PI3K pathway is endogenously activated by the constitutively active Ras family protein ERas (ES cell-expressed Ras). The PI3K pathway utilizes multiple downstream effectors including mTOR (mammalian target of rapamycin), which we have shown to be essential for proliferation in mouse ES cells and early embryos.

Cardiomyocyte differentiation by GATA-4-deficient embryonic stem cells

Development ◽  
1997 ◽  
Vol 124 (19) ◽  
pp. 3755-3764 ◽  
Author(s):  
N. Narita ◽  
M. Bielinska ◽  
D.B. Wilson
Keyword(s):  
Transcription Factor ◽  
In Situ Hybridization ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Cardiomyocyte Differentiation ◽  
Wild Type ◽  
Cell Stage

In situ hybridization studies, promoter analyses and antisense RNA experiments have implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. In this study, we utilized Gata4−/− embryonic stem (ES) cells to determine whether this transcription factor is essential for cardiomyocyte lineage commitment. First, we assessed the ability of Gata4−/− ES cells form cardiomyocytes during in vitro differentiation of embryoid bodies. Contracting cardiomyocytes were seen in both wild-type and Gata4−/− embryoid bodies, although cardiomyocytes were observed more often in wild type than in mutant embryoid bodies. Electron microscopy of cardiomyocytes in the Gata4−/− embryoid bodies revealed the presence of sarcomeres and junctional complexes, while immunofluorescence confirmed the presence of cardiac myosin. To assess the capacity of Gata4−/− ES cells to differentiate into cardiomyocytes in vivo, we prepared and analyzed chimeric mice. Gata4−/− ES cells were injected into 8-cell-stage embryos derived from ROSA26 mice, a transgenic line that expresses beta-galactosidase in all cell types. Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4−/− ES cells contributed to endocardium, myocardium and epicardium. In situ hybridization showed that myocardium derived from Gata4−/− ES cells expressed several cardiac-specific transcripts, including cardiac alpha-myosin heavy chain, troponin C, myosin light chain-2v, Nkx-2.5/Csx, dHAND, eHAND and GATA-6. Taken together these results indicate that GATA-4 is not essential for terminal differentiation of cardiomyocytes and suggest that additional GATA-binding proteins known to be in cardiac tissue, such as GATA-5 or GATA-6, may compensate for a lack of GATA-4.

Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1341-1348 ◽  
Author(s):  
J. Nichols ◽  
E.P. Evans ◽  
A.G. Smith
Keyword(s):  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Leukaemia Inhibitory Factor ◽  
Inhibiting Activity ◽  
Es Cell ◽  
Essential Function ◽  
Germ Line Transmission

The regulatory factor Differentiation Inhibiting Activity/Leukaemia Inhibitory Factor (DIA/LIF) suppresses the differentiation of cultured embryonic stem (ES) cells. In the present study, it is shown that ES cell lines can be derived and maintained in the absence of feeder layers using medium supplemented with purified DIA/LIF. These cells can differentiate normally in vitro and in vivo and they retain the capacity for germ-line transmission. DIA/LIF therefore fulfils the essential function of feeders in the isolation of pluripotential stem cells.

Derivation, Characterization, and In Vitro Differentiation of Canine Embryonic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4059-4059
Author(s):  
Aravind Ramakrishnan ◽  
Brian Hayes ◽  
Sara R. Fagerlie ◽  
Szczepan Baran ◽  
Michael Harkey ◽  
...  
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Clara Cell ◽  
In Vivo Model ◽  
Large Animal ◽  
Es Cell

Abstract Embryonic stem (ES) cells have created considerable excitement in the last few years due to their unlimited potential to produce cells for tissue repair and replacement. However, a large animal pre-clinical model is necessary to establish the safety and efficacy of ES cell-derived tissue replacement therapy. The canine model has long been used in medical research, has been well established to study adult stem cell transplantation and has been highly predictive of clinical outcomes in humans, more so than rodent models. Given the documented record for extrapolating from dog to man, we hypothesize that the dog would serve as an ideal pre-clinical in vivo model for studying the clinical applications of ESC derived tissue. Eleven putative ES cell lines were initiated from canine blastocysts harvested from natural matings. One line described here, FHDO-7, has been maintained through 34 passages and has many characteristics of ES cells from other species. FHDO-7 cells are alkaline phosphatase positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and do not express message for Cdx2 which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miR-302b, miR-302c and miR-367) that have been found to be characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts (MEF) as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders the cells form embryoid bodies (EB). Under various culture conditions the EBs give rise to ectoderm-derived neuronal cells expressing β3-tubulin, mesoderm-derived osteocytes producing bone, and endoderm-derived cells expressing alpha feto protein or Clara cell specific protein. These results indicate that FHDO-7 is a pluripotent embryonic stem cell line.

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