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Observing and Manipulating Pluripotency in Normal and Cloned Mouse Embryos
Abstract
The mouse ooplasm is the ideal platform to study and compare induced and natural pluripotency because it can support both,
after somatic cell nuclear transfer (cloning) and after fertilization, respectively. The amount of pluripotency induced after
cloning is variable but always limited compared to fertilization. It can be visualized conveniently if the nucleus donor cells
carry a green fluorescent protein (GFP) reporter under control of the pluripotency-associated gene Oct4 promoter. Thus we produced cloned and fertilized mouse embryos transgenic for Oct4-GFP (GOF18-∆PE-EGFP). We also developed and validated a live cell imaging method, whereby we resolve and selectively pick cloned embryos that
hold distinct amounts of induced pluripotency as predicted by GFP intensity and measured by embryonic stem cell derivation.
Currently we are developing a microinjection method to change the level of Oct4 without modifying the genome of the embryo.
Here we discuss our findings in relation to the epigenetic reprogramming of the nucleus transplant and to cell fate decisions
in the cloned or fertilized mouse embryo.
KeywordsEmbryo–Embryonic stem cell–Green fluorescent protein–Nuclear transfer–Oct4–Pluripotency–Reprogramming
... Different types of pluripotent cells also exist such as embryonic carcinoma cells and embryonic germ cells. Cells generated via reprogramming are called induced pluripotent stem cells (iPSCs) and do not carry the ethical problems of ESCs because they do not require human embryos (Balbach et al. 2009). ...
Diabetes mellitus (DM) is a chronic, multifactorial metabolic disorder affecting
2–5% of the population and is a major challenge in health. DM type I (T1DM) or
juvenile-onset Diabetes is characterized by autoimmune selective destruction of
insulin-producing pancreatic β cells, which result in an absolute deficiency of insulin
required for glucose metabolism ultimately resulting in the loss of insulin production
and secretion that leads to increase in blood glucose level. So, patients are
dependent on exogenous insulin for their blood glucose control. Usually 60–80% of
the β-cell mass have been destroyed at the time of diagnosis. Insulin replacement
therapy by either insulin pump or multiple daily injections is intensive and often
associated with severe hypoglycemic episodes. Pathogenesis of DM type 2 (T2DM)
is related to genetic, environmental, and lifestyle factors resulting from insulin
resistance in target tissues such as liver, skeletal muscles, and adipose tissues. So, β
cells are unable to sustain the increased demand for insulin, which therefore leads
to chronic hyperglycemia and the onset of T2DM. Both types of diabetes have the
serious long-term complications in different organs such as liver, kidneys, eyes,
heart, nerves, and blood vessels (Diagnosis and classification of diabetes mellitus
2014). Edmonton protocol is the most reliable approach to the treatment of T1DM
by transplantation of whole pancreas or isolated islets. However, the scarcity ofhuman donors and the need for lifelong immunosuppressant to prevent immune
rejection are considered major obstacles to transplantation of islets (Gruessner et al.
2012; Jamiolkowski et al. 2012). Therefore, new therapeutic strategies are needed to
preserve or even promote regeneration of the β-cell mass. Due to the limitation of
using embryonic stem cells (ES) and induced puleripotent stem cells (IPS) in the
clinic, recently cell-based therapy has been focused on mesenchymal stem cells
(MSCs). MSCs have remarkable immunomodulatory properties, and they can be
isolated from adipose tissue and can be differentiated into insulin-producing cells
(IPC). Therefore, they have possible applications in the treatment of type 1 diabetes
(Liu and Han 2008; Wei et al. 2013). In addition, in the past 15 years, a family of
endogenous small noncoding RNAs known as microRNAs (miRNAs) has been discovered
as new players in regulation of protein coding genes. They are a novel class
of endogenous small nc-RNAs, of ~20–30 nucleotides in length that were first discovered
in 1993 in Caenorhabitis elegans and Drosophila and later identified in
many species (Ambros 2004).These nc-RNAs are encoded by up to 3% of all genes,
and approximately 30% of the genes are supposed to be regulated by small RNA
species that regulate gene expression posttranscriptionally (Zhang and Farwell
2008). In mammalians, miRNAs are transcriptional repressor and have inhibitory
effects on RNA stability by base pairing between 3′ untranslated regions (UTRs) of
target mRNAs and miRNA “seed region.” Each miRNA may have multiple targets
and therefor have multiple effects on physiological and pathological processes (van
Rooij 2011). Recently, several miRNAs have been identified that have potential
roles in pancreas development, islet function, insulin secretion, and diabetic complications (Zhang and Farwell 2008; Kantharidis et al. 2011).We also discussed important role of this miRNAs in diabetes and its complications.
... Different types of pluripotent cells also exist such as embryonic carcinoma cells and embryonic germ cells. Cells generated via reprogramming are called induced pluripotent stem cells (iPSCs) and do not carry the ethical problems of ESCs because they do not require human embryos (Balbach et al. 2009). ...
Diabetes mellitus is a group of metabolic disease characterized by insufficient insulin secretion from β cells that usually leads to changes in glucose homeostasis. Autoimmune destruction of β cells in type 1 diabetes and reduced insulin sensitivity in type 2 contribute to diabetes and its complications. Approximately 364 million people are diagnosed with diabetes. Transplantation of whole pancreas or isolated islets could be a cure for diabetes. However, its limited by the scarcity of the donors. In recent years, mesenchymal stem cells have been used for tissue regeneration and opened new clinical avenues for treatment of diabetes mellitus. MSCs, the major stem cells for cell therapy, are therapeutic agent to treat diabetic complications, including diabetic cardiomyopathy diabetic retinopathy, diabetic polyneuropathy, diabetic nephropathy, and diabetic wounds. Another important issue that is provided in this chapter are microRNAs and their important roles in diabetes. miRNAs are a class of small noncoding RNA that are involved in many physiological processes. Distinct modification in blood miRNAs profile is seen in both types of diabetes. So, measurements of the level of specific miRNAs may become useful approaches to identify individuals at risk for developing diabetes mellitus and its complications.
Various experimental or clinical studies in the literature have demonstrated the benefits of stem cells, especially mesenchymal stem cells. This chapter will focus on existing stem cell technologies and their applications in the treatment of wound healing. To understand mechanisms of stem cells in wound healing, we will go over the structure of most commonly wounded tissues, namely, skin, which consist of epithelia and connective tissue. Then we will explain wound healing process with three stages as inflammation, proliferation, and remodeling and the roles of tissue components during wound healing. Treatment of wounds is directly related with the cause, location, and degree of the wound. Therefore, we will describe common wound types with causes under two categories as acute and chronic. We will explain stem cell types which play an important role in tissue regeneration and wound healing in adult life owing to their high competency and self-renewal features. Here, we will give examples of various studies on various wound types with or without underlying diseases. Finally, we will describe recently developed methods such as the use of cells' own products, exosomes, and demonstrate the results of exosome therapies on chronic wounds with most recent examples on human trials.
We have analysed various adult organs and different developmental stages of mouse embryos for the presence of octamer-binding proteins. A variety of new octamer-binding proteins were identified in addition to the previously described Oct1 and Oct2. Oct1 is ubiquitously present in murine tissues, in agreement with cell culture data. Although Oct2 has been described as a B-cell-specific protein, similar complexes were also found with extracts from brain, kidney, embryo and sperm. In embryo and brain at least two other proteins, Oct3 and Oct7, are present. A new microextraction procedure allowed the detection of two maternally expressed octamer-binding proteins, Oct4 and Oct5. Both proteins are present in unfertilized oocytes and embryonic stem cells, the latter containing an additional protein, Oct6. Whereas Oct4 was not found in sperm or testis, it is expressed in male and female primordial germ cells. Therefore Oct4 expression is specific for the female germline at later stages of germ cell development. Our results indicate that a family of octamer-binding proteins is present during mouse development and is differentially expressed during early embryogenesis. Protease clipping experiments of Oct4 and Oct1 suggest that both proteins contain similar DNA-binding domains.
With the advent of human embryonic stem (ES) cell research, many of the paradigms of stem cell biology established by studies in mouse embryos and ES cells, including concepts of lineage restriction, are now being explored in the context of human embryology. The early extraembryonic tissues, trophectoderm and primitive endoderm, are precursor cell lineages that differentiate into tissues essential for the maintenance and patterning of the embryo. In vivo studies using early murine embryos have provided many insights into both the properties and developmental pathways regulating trophectoderm and extraembryonic endoderm differentiation. More recently permanent stem cell lines, trophoblast stem (TS) cells and extraembryonic endoderm (XEN) cells, have been derived from the extraembryonic precursor tissues present in the mouse blastocyst. TS and XEN cells retain many characteristics associated with the in vivo tissue from which they were derived. Analysis of extraembryonic tissues, either in vivo or in vitro, can be used to contrast the properties of embryonic tissues, providing information about discrete stem cell populations within the early embryo and how tissuetissue interactions control differentiation during embryogenesis. Although extraembryonic cell types can be found in the differentiated derivatives of human ES cells, human TS or XEN have yet to be isolated. This chapter outlines our current understanding of human extraembryonic stem cell populations and provides protocols to facilitate their differentiation from human ES cells and subsequent characterization.
The majority of cloned animals derived by nuclear transfer from somatic cell nuclei develop to the blastocyst stage but die after implantation. Mouse embryos that lack an Oct4 gene, which plays an essential role in control of developmental pluripotency, develop to the blastocyst stage and also die after implantation, because they lack pluripotent embryonic cells. Based on this similarity, we posited that cloned embryos derived from differentiated cell nuclei fail to establish a population of truly pluripotent embryonic cells because of faulty reactivation of key embryonic genes such as Oct4 . To explore this hypothesis, we used an in silico approach to identify a set of Oct4 -related genes whose developmental expression pattern is similar to that of Oct4 . When expression of Oct4 and 10 Oct4 -related genes was analyzed in individual cumulus cell-derived cloned blastocysts, only 62% correctly expressed all tested genes. In contrast to this incomplete reactivation of Oct4 -related genes in somatic clones, ES cell-derived cloned blastocysts and normal control embryos expressed these genes normally. Notably, the contrast between expression patterns of the Oct4 -related genes correlated with efficiency of embryonic development of somatic and ES cell-derived cloned blastocysts to term. These observations suggest that failure to reactivate the full spectrum of these Oct4 -related genes may contribute to embryonic lethality in somatic-cell clones.
Adhesion of stem cells - like most cells - is not just a membrane
phenomenon. Most tissue cells need to adhere to a ``solid'' for
viability, and over the last decade it has become increasingly clear
that the physical ``elasticity'' of that solid is literally ``felt'' by
cells. Here we show that Mesenchymal Stem Cells (MSCs) specify lineage
and commit to phenotypes with extreme sensitivity to the elasticity
typical of tissues [1]. In serum only media, soft matrices that mimic
brain appear neurogenic, stiffer matrices that mimic muscle are
myogenic, and comparatively rigid matrices that mimic collagenous bone
prove osteogenic. Inhibition of nonmuscle myosin II activity blocks all
elasticity directed lineage specification, which indicates that the
cytoskeleton pulls on matrix through adhesive attachments. Results have
significant implications for `therapeutic' stem cells and have motivated
development of a proteomic-scale method to identify mechano-responsive
protein structures [2] as well as deeper physical studies of matrix
physics [3] and growth factor pathways [4]. [4pt] [1] A. Engler, et al.
Matrix elasticity directs stem cell lineage specification. Cell
(2006).[0pt] [2] C.P. Johnson, et al. Forced unfolding of proteins
within cells. Science (2007).[0pt] [3] A.E.X. Brown, et al. Multiscale
mechanics of fibrin polymer: Gel stretching with protein unfolding and
loss of water. Science (2009).[0pt] [4] D.E. Discher, et al. Growth
factors, matrices, and forces combine and control stem cells. Science
(2009).
The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being1, 2. ES cell lines are conventionally isolated from the inner cell mass of blastocysts3, 4, 5 and, in a few instances, from cleavage stage embryos6, 7, 8, 9. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines—using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects10—that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.
Abramoff, M.D., Magelhaes, P.J., Ram, S.J. "Image Processing with ImageJ". Biophotonics International, volume 11, issue 7, pp. 36-42, 2004.
Explore the potential for nanotechnologies to transform future mobile and Internet communications. Based on a research collaboration between Nokia, Helsinki University of Technology, and the University of Cambridge, here leading researchers review the current state-of-the art and future prospects for: • Novel multifunctional materials, dirt repellent, self-healing surface materials, and lightweight structural materials capable of adapting their shape • Portable energy storage using supercapacitor-battery hybrids based on new materials including carbon nanohorns and porous electrodes, fuel cell technologies, energy harvesting and more efficient solar cells • Electronics and computing advances reaching beyond IC scaling limits, new computing approaches and architectures, embedded intelligence and future memory technologies. • Nanoscale transducers for mechanical, optical and chemical sensing, sensor signal processing, and nanoscale actuation • Nanoelectronics to create ultrafast and adaptive electronics for future radio technologies • Flat panel displays with greater robustness, improved resolution, brightness and contrast, and mechanical flexibility • Manufacturing and innovation processes, plus commercialization of nanotechnologies.
The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13(GFPneo) embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13(lacZneo) embryos. Similarly, Hoxa1(GFPneo) expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1(GFPneo) embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm. journal article
Blastomeres isolated from 4- and 8-cell mouse eggs were inserted into empty zonae and transferred to the oviduct. The products of both types of blastomere were capable of inducing decidual formation. One implant produced by an isolated blastomere from a 4-cell egg contained a small, retarded embryo at 5 1/2 days but most decidua from blastomeres of either 4- or 8-cell eggs contained only a few trophoblast giant cells. It is suggested that this lack of totipotency is due to insufficient cells being present at cavitation rather than restriction in developmental potential.