LIF, the ES-cell inhibition factor, reversibly blocks nephrogenesis in cultured mouse kidney rudiments

Development ◽  
1991 ◽  
Vol 113 (1) ◽  
pp. 193-198 ◽  
Author(s):  
J.B. Bard ◽  
A.S. Ross
Keyword(s):  
Embryonic Stem ◽  
Mouse Kidney ◽  
Leukaemia Inhibitory Factor ◽  
Es Cell ◽  
Metanephric Mesenchyme ◽  
Ureteric Bud ◽  
Cell Inhibition ◽  
Striking Effect

Mouse kidney induction proceeds in vitro much as it does in vivo: the ureteric bud bifurcates to give collecting ducts while the mesenchyme condenses into aggregates which epithelialise and then elongate into tubules with glomerular and other nephron structures. We report here that the factor known as LIF (leukaemia inhibitory factor), which regulates the differentiation and growth of embryonic-stem (ES) and other cells in culture, has little effect in vitro on growth or on ureteric-bud morphogenesis other than to stimulate the bifurcation process. It does however exert a striking effect on the mesenchyme. At about four times the concentration required to inhibit ES-cell differentiation, LIF strongly but reversibly blocks the effects of metanephric mesenchyme induction: although mesenchyme condenses around growing duct tips, the number of mature nephrons that form over 6 days is reduced by 75% or more. The few nephrons that do develop in the presence of LIF probably come from mesenchyme already induced at the time of culture and are indistinguishable from those that form in controls as assayed by morphology, by X-gal staining of endogenous galactosidase and by antibodies to brush-border and CD15 antigens. There is a further unexpected feature of rudiments cultured in LIF which is absent in controls: they contain an unexpectedly high number of stable epithelialised aggregates that express laminin around their periphery and which do not develop further. These results argue that the process of nephrogenesis involves at least two distinct stages which can be blocked by LIF: the effect of the initial induction and the future development of epithelialised aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The homeodomain protein Nanog and pluripotency in mouse embryonic stem cells

2005 ◽  
Vol 33 (6) ◽  
pp. 1518-1521 ◽  
Author(s):  
A. Yates ◽  
I. Chambers
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Leukaemia Inhibitory Factor ◽  
Homeodomain Protein ◽  
Es Cell ◽  
Homeodomain Proteins ◽  
Morphogenetic Protein

Intrinsic regulators of the pluripotency of mouse ES (embryonic stem) cells include the homeodomain proteins Oct4 and the recently identified Nanog. When overexpressed, Nanog displays the unique attribute of robustly sustaining ES cell self-renewal in the absence of the otherwise requisite extracellular stimulation by LIF (leukaemia inhibitory factor) and BMP (bone morphogenetic protein). Here, we review our current understanding of the function of Nanog in pluripotent stem cells both in vitro and in vivo.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2273-2282 ◽  
Author(s):  
W. Dean ◽  
L. Bowden ◽  
A. Aitchison ◽  
J. Klose ◽  
T. Moore ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Upstream Region ◽  
Imprinted Genes ◽  
Epigenetic Alterations ◽  
Es Cell ◽  
Biallelic Expression

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

scholarly journals 173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION

2005 ◽  
Vol 17 (2) ◽  
pp. 237 ◽  
Author(s):  
G. Lazzari ◽  
I. Lagutina ◽  
G. Crotti ◽  
P. Turini ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Cell Mass ◽  
Embryonic Stem ◽  
Farm Animals ◽  
Es Cell ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
In Vivo Culture

Attempts to derive true embryonic stem cells in large farm animals rely on the supply of good quality embryos. In these species, including the pig, pre-implantation-stage embryos can be produced by in vitro techniques from slaughterhouse ovaries. The objective of this study was to evaluate the ability of the inner cell masses (ICMs) of pig embryos, produced in vitro by different methods, to provide viable initial outgrowths of ICM cells that could be subsequently subcultured and expanded. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40–44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng/mL long-EGF, 100 ng/mL long-IGF1, 5 ng/mL bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen-thawed semen was separated on a percoll gradient and diluted in TALP medium with PHE (penicillamine, hypotaurine, epinefrine) to a concentration ranging from 0.05 to 0.1 million sperm per mL. Oocytes were partially decumulated, co-incubated with sperm for 24 h, and finally denuded and cultured in microdrops of mSOFaa or NCSU. After cleavage, approximately half of the cleaved embryos were surgically transferred into the sheep oviduct for 4 days of in vivo culture and the remaining embryos were left in vitro in the two media. On Day +6 in vivo-cultured embryos were recovered from the sheep oviduct. Blastocyst formation and quality were comparatively evaluated in the three culture groups. Quality specifically referred to the morphology/size of the ICM according to the following criteria: ICM A (large/prominent), ICM B (flat), and ICM C (non-visible). All embryos with a visible inner cell mass were subjected to microdissection with needles to recover the ICMs that were then plated on feeder-layers of mitomycin-treated STO fibroblasts. Attachment and outgrowth was evaluated 48–72 h post-plating. Results are presented in Table 1. Our data indicate that in vivo culture of pig embryos in the sheep oviduct greatly enhance both blastocyst development and ICM quality. As a consequence the efficiency of outgrowth formation, following plating for ES cell derivation, was significantly higher with ICMs derived from IVM-IVF pig embryos cultured in vivo as compared to their in vitro-cultured counterparts. Within the two culture media tested for in vitro culture, SOF and NCSU, the rate of blastocyst formation was similar but the quality of SOF-cultured embryos is higher. In conclusion, embryo/ICM quality represents a fundamental requirement for the derivation of ES cell lines, and in vivo culture in the sheep oviduct provides the most efficient source of high quality IVM-IVF pig embryos. Table 1. Blastocyst development and ICM quality of in vitro-produced pig embryos This work was supported by the Istituto Superiore di Sanità, Programma Nazionale Cellule Staminali, Rome, Italy, grant No. CS 11.

GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4108-4118 ◽  
Author(s):  
Naruyoshi Suwabe ◽  
Satoru Takahashi ◽  
Toru Nakano ◽  
Masayuki Yamamoto
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Es Cell ◽  
Globin Mrna ◽  
Differentiated Cells ◽  
Vitro Analysis ◽  
Definitive Hematopoiesis

Abstract Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells by Their Genetic Manipulation Using the Third Generation Lentiviral Vector.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 495-495
Author(s):  
Ryo Kurita ◽  
Erika Sasaki ◽  
Takashi Hiroyama ◽  
Tomoko Yokoo ◽  
Yukoh Nakazaki ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Preclinical Studies ◽  
Es Cell ◽  
Hematopoietic Stem

Abstract Since the successful establishment of human embryonic stem (ES) cell lines in 1998, transplantation of differentiated ES cells to specific organ has been expected to complete its defective function. For the realistic medicine, the preclinical studies using animal model systems including non-human primates are essential. We have already demonstrated that non-human primates of common marmosets (CM) are suitable for the laboratory animal models for preclinical studies of hematopoietic stem cell therapy. In this study, we investigated the in vitro and in vivo differentiation of CM ES cells to hematopoietic cells by exogenous gene transfer methods in order to study the feasibility of future gene modified ES cell therapy. First, we tried various in vitro culture conditions including systems using embryoid bodies or co-culturing with stromal cells to induce hematopoietic cells, but the frequency of inducing hematopoietic cells was very low. The expression of CD45 and gata1 could not be detected in both conditions, suggesting that our culture conditions were incomplete for induction of hematopoietic cells from CM ES cells. Next we examined gene transduction methods by using VSV-G pseudotyped human immunodeficiency virus (HIV) vectors. We constructed the HIV vectors containing hematopoietic genes such as tal1/scl, gata1, gata2, hoxB4 and Lh2 genes under the EF1a promoter and transduced them into CM ES cells. Only in the case of tal1/scl overexpression, not other genes, hematopoietic induction from CM ES cells was dramatically increased and multi-lineage blood cells consisting of erythroid cells, granulocytes, macrophages and megakaryocytes, were confirmed by immunochemical and morphological analyses. Furthermore, RT-PCR results showed that several hematopoietic marker genes including CD34 were expressed higher in the tal1/scl overexpressed ES-derived cells. After the xenotransplantation of ES-derived cells into the immunodeficient mice, CM CD45+ cells and immature erythroids and megakaryocytic cells were observed only in the ES-tal1-injected mice, indicating that enforced expression of tal1/scl into ES cells led to highly efficient hematopoietic cell differentiation in vivo. Taken together, it was suggested that the transduction of exogenous tal1/scl cDNA into ES cells by HIV vector was the promising method for the efficient differentiation from CM ES cells to hematopoietic stem cells. Further examinations are required to determine the long-term hematopoietic reconstitute capacity and the safety of the tal1/scl transduced ES cells in marmoset for the purpose of developing new hematopoietic stem cell therapy.

scholarly journals Human embryonic stem cells for brain repair?

2007 ◽  
Vol 363 (1489) ◽  
pp. 87-99 ◽  
Author(s):  
Su-Chun Zhang ◽  
Xue-Jun Li ◽  
M Austin Johnson ◽  
Matthew T Pankratz
Keyword(s):  
Stem Cells ◽  
Motor Neurons ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dopamine Neurons ◽  
Neural Cells ◽  
Es Cell ◽  
Human Es Cells

Cell therapy has been perceived as the main or ultimate goal of human embryonic stem (ES) cell research. Where are we now and how are we going to get there? There has been rapid success in devising in vitro protocols for differentiating human ES cells to neuroepithelial cells. Progress has also been made to guide these neural precursors further to more specialized neural cells such as spinal motor neurons and dopamine-producing neurons. However, some of the in vitro produced neuronal types such as dopamine neurons do not possess all the phenotypes of their in vivo counterparts, which may contribute to the limited success of these cells in repairing injured or diseased brain and spinal cord in animal models. Hence, efficient generation of neural subtypes with correct phenotypes remains a challenge, although major hurdles still lie ahead in applying the human ES cell-derived neural cells clinically. We propose that careful studies on neural differentiation from human ES cells may provide more immediate answers to clinically relevant problems, such as drug discovery, mechanisms of disease and stimulation of endogenous stem cells.

scholarly journals Genetic Dissection of Cadherin Function during Nephrogenesis

2002 ◽  
Vol 22 (5) ◽  
pp. 1474-1487 ◽  
Author(s):  
Ulf Dahl ◽  
Anders Sjödin ◽  
Lionel Larue ◽  
Glenn L. Radice ◽  
Stefan Cajander ◽  
...  
Keyword(s):  
Kidney Development ◽  
Morphological Changes ◽  
Genetic Dissection ◽  
Metanephric Mesenchyme ◽  
Ureteric Bud ◽  
Metanephric Kidney ◽  
The Individual ◽  
Deficient Mice

ABSTRACT The distinct expression of R-cadherin in the induced aggregating metanephric mesenchyme suggests that it may regulate the mesenchymal-epithelial transition during kidney development. To address whether R-cadherin is required for kidney ontogeny, R-cadherin-deficient mice were generated. These mice appeared to be healthy and were fertile, demonstrating that R-cadherin is not essential for embryogenesis. The only kidney phenotype of adult mutant animals was the appearance of dilated proximal tubules, which was associated with an accumulation of large intracellular vacuoles. Morphological analysis of nephrogenesis in R-cadherin −/− mice in vivo and in vitro revealed defects in the development of both ureteric bud-derived cells and metanephric mesenchyme-derived cells. First, the morphology and organization of the proximal parts of the ureteric bud epithelium were altered. Interestingly, these morphological changes correlated with an increased rate of apoptosis and were further supported by perturbed branching and patterning of the ureteric bud epithelium during in vitro differentiation. Second, during in vitro studies of mesenchymal-epithelial conversion, significantly fewer epithelial structures developed from R-cadherin −/− kidneys than from wild-type kidneys. These data suggest that R-cadherin is functionally involved in the differentiation of both mesenchymal and epithelial components during metanephric kidney development. Finally, to investigate whether the redundant expression of other classic cadherins expressed in the kidney could explain the rather mild kidney defects in R-cadherin-deficient mice, we intercrossed R-cadherin −/− mice with cadherin-6−/− , P-cadherin −/−, and N-cadherin +/− mice. Surprisingly, however, in none of the compound knockout strains was kidney development affected to a greater extent than within the individual cadherin knockout strains.

The homeoprotein Hex is required for hemangioblast differentiation

Blood ◽  
2003 ◽  
Vol 102 (7) ◽  
pp. 2428-2435 ◽  
Author(s):  
Ying Guo ◽  
Rebecca Chan ◽  
Heather Ramsey ◽  
Weiming Li ◽  
Xiaodong Xie ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Chimeric Mouse ◽  
Es Cell ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Temporal And Spatial

Abstract The first hematopoietic and endothelial progenitors are derived from a common embryonic precursor termed the hemangioblast. The genetic cascades that regulate the differentiation of the hemangioblast to hematopoietic and endothelial cells are largely unknown. In general, much of embryonic development is coordinately regulated by temporal and spatial expression of transcription factors, such as the Homeobox (Hox) gene family. We and others isolated a divergent homeobox gene termed Hex (or Prh) that is preferentially expressed in hematopoietic and endothelial cells. Using in vitro Hex-/- embryonic stem (ES) cell differentiation, in vivo yolk sac hematopoietic progenitor assays, and chimeric mouse analysis, we found that Hex is required for differentiation of the hemangioblast to definitive embryonic hematopoietic progenitors and to a lesser extent endothelial cells. Therefore, Hex is a novel regulator of hemangioblast differentiation to hematopoietic and endothelial cells. (Blood. 2003;102:2428-2435)

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