Study Reveals the Genetic Start-up of a Human Embryo

Study Reveals the Genetic Start-up of a Human Embryo

Thursday, 03 September 2015

An international team of scientists led from Sweden’s Karolinska Institutet has for the first time mapped all the genes that are activated in the first few days of a fertilised human egg. The study, which is being published in the journal Nature Communications, provides an in-depth understanding of early embryonic development in human – and scientists now hope that the results will help finding for example new therapies against infertility.

At the start of an individual’s life there is a single fertilised egg cell. One day after fertilisation there are two cells, after two days four, after three days eight and so on, until there are billions of cells at birth. The order in which our genes are activated after fertilisation has remained one of the last uncharted territories of human development.

Juha Kere is a Professor of Molecular Genetics at

Karolinska Institutet. Credit: Ulf Sirborn.

There are approximately 23,000 human genes in total. In the current study, scientists found that only 32 of these genes are switched on two days after fertilization, and by day three there are 129 activated genes. Seven of the genes found and characterised had not been discovered previously.

“These genes are the ‘ignition key’ that is needed to turn on human embryonic development. It is like dropping a stone into water and then watching the waves spread across the surface”, says principal investigator Juha Kere, professor at theDepartment of Biosciences and Nutrition at Karolinska Institutet and also affiliated to the SciLifeLab facility in Stockholm.

The researchers had to develop a new way of analysing the results in order to find the new genes. Most genes code for proteins but there are a number of repeated DNA sequences that are often considered to be so-called ‘junk DNA’, but are in fact important in regulating gene expression.

Treatment of infertility

In the current study, the researchers show that the newly identified genes can interact with the ‘junk DNA’, and that this is essential to the start of development.

Outi Hovatta is a Professor of Obstetrics and

Gynaecology at Karolinska Institutet. Credit:

Ulf Sirborn.

“Our results provide novel insights into the regulation of early embryonic development in human. We identified novel factors that might be used in reprogramming cells into so-called pluripotent stem cells for possible treatment of a range of diseases, and potentially also in the treatment of infertility”, says Outi Hovatta, professor at Karolinska Institutet’s Department of Clinical Science, Intervention and Technology, and a senior author.

The study was a collaboration between three research groups from Sweden and Switzerland that each provided a unique set of skills and expertise. The work was supported by the Karolinska Institutet Distinguished Professor Award, the Swedish Research Council, the Strategic Research Program for Diabetes funding at Karolinska Institutet, Stockholm County, the Jane & Aatos Erkko Foundation, the Instrumentarium Science Foundation, and the Åke Wiberg and Magnus Bergvall foundations. The computations were performed on resources provided by SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX).

Source: Karolinska Institutet

Contact: KI Press Office

Reference:

Novel PRD-like homeodomain transcription factors and retrotransposon elements in early human development

Virpi Töhönen, Shintaro Katayama, Liselotte Vesterlund, Eeva-Mari Jouhilahti, Mona Sheikhi, Elo Madissoon, Giuditta Filippini-Cattaneo, Marisa Jaconi, Anna Johnsson, Thomas R. Bürglin, Sten Linnarsson, Outi Hovatta and Juha Kere

Nature Communications, 3 September 2015, doi: 10.1038/NCOMMS9207

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From Pluripotency to Totipotency

Scientists discover mechanism that may lead to more efficient reprogramming of somatic cells

Wednesday, 05 August 2015

Human embryonic stem cells have the potential

to form in vitro neural tube-like structures of the

embryo. Credit: Inserm/Benchoua Alexandra.

While it is already possible to obtain in vitro pluripotent cells (i.e., cells capable of generating all tissues of an embryo) from any cell type, researchers from Maria-Elena Torres-Padilla's team from Institut de Génétique et de Biologie Moléculaire et Cellulaire, have pushed the limits of science even further. They managed to obtain totipotent cells with the same characteristics as those of the earliest embryonic stages and with even more interesting properties. Obtained in collaboration with Juanma Vaquerizas from the Max Planck Institute for Molecular Biomedicine (Münster, Germany), these results are published on 3rd of August in the journal Nature Structural & Molecular Biology.

Totipotency vs pluripotency

Just after fertilization, when the embryo is comprised of only 1 or 2 cells, cells are "totipotent", that is to say, capable of producing an entire embryo as well as the placenta and umbilical cord that accompany it. During the subsequent rounds of cell division, cells rapidly lose this plasticity and become "pluripotent". At the blastocyst stage (about thirty cells), the so-called "embryonic stem cells" can differentiate into any tissue, although they alone cannot give birth to a foetus anymore. Pluripotent cells then continue to specialise and form the various tissues of the body through a process called cellular differentiation.

For some years, it has been possible to re-programme differentiated cells into pluripotent ones, but not into totipotent cells. Now, the team of Maria-Elena Torres-Padilla has studied the characteristics of totipotent cells of the embryo and found factors capable of inducing a totipotent-like state.

“Totipotency is a much more flexible state than the pluripotent state and its potential applications are extraordinary”, says Maria-Elena Torres-Padilla, who led the study.

Looking for the keys of totipotency

When culturing pluripotent stem cells in vitro, a small amount of totipotent cells appear spontaneously; these are called "2C-like cells" (named after their resemblance to the 2-cell stage embryo). The researchers compared these cells to those present in early embryos in order to find their common characteristics and those that make them different from pluripotent cells. In particular, the teams found that the DNA was less condensed in totipotent cells and that the amount of the protein complex CAF1 was diminished. A closer look revealed that CAF1 – already known for its role in the assembly of chromatin (the organised state of DNA) – is responsible for maintaining the pluripotent state by ensuring that the DNA is wrapped around histones. Based on this hypothesis, the Torres-Padilla team was able to induce a totipotent state by inactivating the expression of the CAF1 complex, which led to chromatin reprogramming into a less condensed state.

A 2C-like cell (green) is different from an

embryonic stem cell (magenta). Credit:

IGBMC/Maria-Elena Torres-Padilla.

In order to carefully examine at a molecular level the similarities between 2-cell stage embryos, 2C-like cells and those induced by inactivating the CAF1 complex, the Torres-Padilla team then joined forces with the Vaquerizas laboratory to analyse, in a genome-wide fashion, the gene expression programmes of these cells. The scientists found that the induced, CAF1-depleted, totipotent cells overexpressed a significant amount of 2-cell stage embryo genes.

“One could imagine that if cells lose their ability to assemble chromatin, this would affect gene expression”, explains Cells-in-Motion PhD student Rocio Enriquez-Gasca of Juanma Vaquerizas’ lab, who performed the computational analyses of the work.

“So it was really exciting to realise that the resulting gene expression programme in fact significantly overlaps with that of early embryo, totipotent cells”.

Moreover, the teams found that specific classes of repetitive elements (repeated sequences of DNA that form around 50% of the mouse and human genomes) were also up-regulated in induced totipotent-like cells, a hallmark of the 2-cell embryo.

“The computational analysis of expression of repetitive elements is very challenging, since these are found many times in the genome”, says Juanma Vaquerizas.

“Now it is key to understand why these repetitive elements and gene expression programmes are both up-regulated in totipotent cells”.

These results provide new elements for the understanding of pluripotency and could increase the efficiency of reprogramming somatic cells to be used for applications in regenerative medicine.

Source: INSERM

Contact: Maria-Elena Torres-Padilla 

Reference:

Early embryonic-like cells are induced by down-regulation of replication-dependent chromatin assembly

Takashi Ishiuchi, Rocio Enriquez-Gasca, Eiji Mizutani, Ana Boškovi, Celine Ziegler-Birling, Diego Rodriguez-Terrones, Teruhiko Wakayama, Juan M. Vaquerizas & Maria-Elena Torres-Padilla

Nature Structural & Molecular Biology, 3 Aug 2015, doi:10.1038/nsmb.3066

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Researchers Reconstruct Early Stages of Embryo Development

Researchers Reconstruct Early Stages of Embryo Development

Tuesday, 04 November 2014

Researchers at the University of Cambridge have managed to reconstruct the early stage of mammalian development using embryonic stem cells, showing that a critical mass of cells – not too few, but not too many – is needed for the cells to being self-organising into the correct structure for an embryo to form.

All organisms develop from embryos: a cell divides generating many cells. In the early stages of this process, all cells look alike and tend to aggregate into a featureless structure, more often than not a ball. Then, the cells begin to 'specialise' into different types of cell and space out asymmetrically, forming an axis which begins to provide a structure for the embryo to develop along.

In animal embryos this stage is followed by a process known as gastrulation: a choreographed movement of the cells that, using the initial axis as a reference, positions the head and the tail, the front and the back. During the process, the cells begin to form three distinct layers: the endoderm, mesoderm and ectoderm, determining which tissues or organs the cells will then develop into.

Professor Alfonso Martinez-Arias from the Department of Genetics at the University of Cambridge, who led the research, says:

"Gastrulation was described by biologist Professor Lewis Wolpert as being 'truly the most important event in your life' because it creates the blueprint of an organism. Axis formation and gastrulation are the two central processes that initiate the development of an organism and are inextricably associated with the embryo. We have managed to recreate this for the first time in the lab."

Professor Martinez-Arias and colleagues, supported by the European Research Council and the Wellcome Trust, have reconstructed these early stages of development using mouse embryonic stem cells. Embryonic stem cells, discovered in the Department of Genetics in the 1980s (for which Sir Martin Evans was awarded the Nobel Prize in Physiology or Medicine 2007), have become an important tool for developmental biology, understanding disease, and in regenerative medicine due to the ability to give rise to all cell types in culture. Over the last few years, they have been used to 'grow' organs including the eye and the cerebral cortex; surprisingly, these structures develop without an axis.

In research published today in the journal Development, the researchers report a way to coax cells to reorganize in the manner that they do in an embryo, creating an axis and undergoing movements and organisations that mimic the process of gastrulation. Over the years researchers have been making aggregates of embryonic stem cells to obtain certain cell types, for example red blood cells. However, these aggregates lack structure and the different cell types emerge in a disorganised fashion. This is the first time that researchers have been able to elicit axis formation, spatial organisation and gastrulation-like movements from aggregates of embryonic stem cells.

The researchers show that if the number of cells aggregated initially is similar to that of a mouse embryo, the cells generate a single axis and this serves as a template for a sequence of events that mimics those of the early embryo. By manipulating the signals that the cells see at a particular time, the researchers were able to influence what type of cell they become and how they are organised. In one of the experiments, for example, activation of a particular signal at the correct time elicits the appearance of the mesoderm, endoderm and ectoderm – the precursors of all cell types – with a spatial organization similar to that of an embryo.

Using this experimental system, the researchers were able to generate the early stages of a spinal cord, which they showed forms as part of the process of gastrulation. This finding complements previous research from the University of Edinburgh and the National Institute for Medical Research which showed that embryonic stem cells can be coaxed into this spinal cord cells; however, the Cambridge researchers showed that in the embryo-like aggregates, the structural organization is more robust and allows for the polarised growth of the tissue.

Professor Martinez-Arias adds:

"It is early days but this system promises insights into the early stages of development and what determines the specification of the different cell types. This will allow more robust protocols for differentiation with cues that mimic those that the cells are subject to in embryos.”

"Most significantly, the system will provide a means to test, experimentally, how a homogeneous group of cells organizes itself in space, a central process in the development of any organism, and the ability to recreate in culture the niches that adult stem cells create during embryogenesis and which have remained elusive experimentally."

Source: University of Cambridge

Contact: Craig Brierley

References:

Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse ES cells

Susanne C. van den Brink, Peter Baillie-Johnson, Tina Balayo, Anna-Katerina Hadjantonakis, Sonja Nowotschin, David A. Turner, and Alfonso Martinez Arias

Development 2014 141:4231-4242; doi:10.1242/dev.113001

Wnt/β-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse ES cells

David A. Turner, Penelope C. Hayward, Peter Baillie-Johnson, Pau Rué, Rebecca Broome, Fernando Faunes, and Alfonso Martinez Arias

Development; 2014 141:4243-4253; doi:10.1242/dev.112979

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Researchers Create 'Naïve' Pluripotent Human Embryonic Stem Cells

Researchers Create 'Naïve' Pluripotent Human Embryonic Stem Cells

Saturday, 26 July 2014

Phase and fluorescence images of conventional

(primed) human embryonic stem cells (ESCs)

and naïve human ESCs generated in the

presence of 5 small molecule inhibitors.

The naïve human ESCs exhibit activation of a

fluorescent reporter linked to an enhancer of

theOCT4 gene that is specifically used in

the naïve state. 40X magnification. Credit:

Courtesy of Thorold Theunissen. 

For years, researchers and patients have hoped that embryonic stem cells (ESCs) — capable of forming nearly any cell type in the body — could provide insight into numerous diseases perhaps even be used to treat them. Yet progress has been hampered by the inability to transfer research and tools from mouse ESC studies to their human counterparts, in part because human ESCs are "primed" and slightly less plastic than the mouse cells.

Now Thorold Theunissen, Benjamin Powell, and Haoyi Wang, who are scientists in the lab of Whitehead Institute Founding Member Rudolf Jaenisch, have discovered how to manipulate and maintain human ESCs in a "naïve" or base pluripotent state similar to that of mouse ESCs without the use of any reprogramming factors. Their work is described in this week's issue of the journal Cell Stem Cell.

Naïve mice ESCs are well-studied, and scientists have a strong understanding of how they function and mature into more specialized cells. But this understanding is of limited use in human ESC research, as the human cells look different, grow differently, and rely on different genes than mouse ESCs. According to Theunissen, the disparities between mouse and human ESCs are attributable not to species-specific differences but rather to differences of cell state.

In naïve mouse ESCs, a particular enhancer of the gene OCT4 is active, prompting the researchers to look for the presence of this marker as a means to identify rare naïve human ESCs. With this unbiased reporter system in hand, the Jaenisch team determined that a cocktail of five small molecules with a few additional growth factors can induce and support the conversion of primed human ESCs to a naïve state with or without using reprogramming factors to jumpstart the process.

By applying this cocktail to human blastocysts, the scientists could also isolate naïve human stem cells.

"This is important because if this cocktail only works in existing lines of human ESCs, you might wonder, does this really capture a distinct state or is this artificial?" says Theunissen.

"Since the cocktail works directly on human blastocysts, I think it suggests that we're really capturing a cell state that is already present in the early human embryo."

Although other labs have recently reported creating naïve human ESCs, Theunissen, Powell, and Wang question these results as the cells produced through these techniques lack the gene expression and epigenetic profiles of naïve human ESCs. Yet, the Jaenisch lab believes they have now finally unlocked a way to create and maintain this important type of cell and are looking forward to exploring its potential.

"We have discovered a new pathway to generate something we believe is a totally different state of pluripotency in human ESCs that is very close to the mouse naïve state," says Jaenisch, who is also a professor of biology at MIT.

"These cells may be essential for ESC technology, and that is an area we're looking forward to investigating. Now the big question for us is, does this state exist in vivo in embryos? Right now, we don't know, and that is a very interesting line of research."

Source: Whitehead Institute for Biomedical Research 

Contact: Nicole Giese Rura

Reference:

Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency

Thorold W. Theunissen, Benjamin E. Powell, Haoyi Wang, Maya Mitalipova, Dina A. Faddah, Jessica Reddy, Zi Peng Fan, Dorothea Maetzel, Kibibi Ganz, Linyu Shi, Tenzin Lungjangwa, Sumeth Imsoonthornruksa, Yonatan Stelzer, Sudharshan Rangarajan, Ana D'Alessio, Jianming Zhang, Qing Gao, Meelad M. Dawlaty, Richard A. Young, Nathanael S. Gray, and Rudolf Jaenisch

Cell Stem Cell, July 24, 2014, http://www.cell.com/cell-stem-cell/abstract/S1934-5909(14)00298-7

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Reprogramming in situ: Spanish Team is First to Produce Embryonic Stem Cells in Living Adult Organisms

The characteristics of the obtained stem cells correspond to a primitive totipotent state that has never before been obtained

Wednesday, 11 September 2013

A team from the Spanish National Cancer Research Centre (CNIO) has become the first to make adult cells from a living organism retreat in their evolutionary development to recover the characteristics of embryonic stem cells.

Pictured are Manuel Serrano and Maria Abad

in his laboratory at the CNIO. Credit: Spanish

National Cancer Research Center (CNIO).

Researchers have also discovered that these embryonic stem cells, obtained directly from the inside of the organism, have a broader capacity for differentiation than those obtained via in vitro culture. Specifically, they have the characteristics of totipotent cells: a primitive state never before obtained in a laboratory.

The study, carried out by CNIO, was led by Manuel Serrano, the director of the Molecular Oncology Programme and head of the Tumoural Suppression Laboratory. The study was supported by Manuel Manzanares's team from the Spanish National Cardiovascular Research Centre (CNIC).

Embryonic stem cells are the main focus for the future of regenerative medicine. They are the only ones capable of generating any cell type from the hundreds of cell types that make up an adult organism, so they are the first step towards curing illnesses such as Alzheimer, Parkinson's disease or diabetes. Nevertheless, this type of cell has a very short lifespan, limited to the first days of embryonic development, and they do not exist in any part of an adult organism.

One of the greatest achievements in recent biomedical research was in 2006 when Shinya Yamanaka managed to create embryonic stem cells (pluripotent stem cells, induced in vitro or in vitro iPSCs) in a laboratory from adult cells, via a cocktail of just four genes. Yamanaka's discovery, for which he was awarded the Nobel Prize in Medicine in 2012, opened a new horizon in regenerative medicine.

CNIO researchers have taken another step forward, by achieving the same as Yamanaka, but this time within the same organism, in mice, without the need to pass through in vitro culture dishes. Generating these cells within an organism brings this technology even closer to regenerative medicine.

The first challenge for CNIO researchers was to reproduce the Yamanaka experiment in a living being. They chose a mouse as a model organism. Using genetic manipulation techniques, researchers created mice in which Yamanaka's four genes could be activated at will. When these genes were activated, they observed that the adult cells were able to retreat in their evolutionary development to become embryonic stem cells in multiple tissues and organs.

María Abad, the lead author of the article and a researcher in Serrano's group, said:

"This change of direction in development has never been observed in nature. We have demonstrated that we can also obtain embryonic stem cells in adult organisms and not only in the laboratory".

Manuel Serrano added that:

"We can now start to think about methods for inducing regeneration locally and in a transitory manner for a particular damaged tissue".

Stem cells obtained in mice also show totipotent characteristics never generated in a laboratory, equivalent to those present in human embryos at the 72-hour stage of development, when they are composed of just 16 cells.

In comparison with the cells obtained with the technique developed by Yamanaka, the stem cells obtained by CNIO therefore represent an even earlier embryonic state, with greater capacity for differentiation.

The authors were even able to induce the formation of pseudo-embryonic structures (teratomas) in the thoracic and abdominal cavities of the mice. These pseudo-embryos displayed the three layers typical of embryos (ectoderm, mesoderm and endoderm), and extra-embryonic structures such as the Vitelline membrane and even signs of blood cell formation.

"This data tell us that our stem cells are much more versatile than Yamanaka's in vitro iPSCs, whose potency generates the different layers of the embryo but never tissues that sustain the development of a new embryo, like the placenta", said the CNIO researcher.

The authors emphasise that the possible therapeutic applications of their work are still distant, but they admit that, without doubt, it might mean a change of direction for stem cell research, for regenerative medicine or for tissue engineering.

"Our stem cells also survive outside of mice, in a culture, so we can also manipulate them in a laboratory", said Abad.

"The next step is studying if these new stem cells are capable of efficiently generating different tissues such as that of the pancreas, liver or kidney".

Source: Centro Nacional de Investigaciones Oncologicas (CNIO) 

Contact: Juan J. Gomez

Reference:

Reprogramming in vivo produces teratomas and iPSCs with totipotency features

María Abad, Lluc Mosteiro, Cristina Pantoja, Marta Cañamero, Teresa Rayón, Inmaculada Ors, Osvaldo Graña, Diego Megías, Orlando Domínguez, Dolores Martínez, Miguel Manzanares, Sagrario Ortega, Manuel Serrano

Nature (2013), DOI: 10.1038/nature12586

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Rewinding Development

A step forward for stem cell research

Thursday, 06 June 2013

Scientists at the Danish Stem Cell Center, DanStem, at the University of Copenhagen have discovered that they can make embryonic stem cells regress to a stage of development where they are able to make placenta cells as well as the other foetal cells. This significant discovery, published in the journal Cell Reports today, has the potential to shed new light on placenta related disorders that can lead to problematic pregnancies and miscarriages.

The picture shows a 9.5-day-old mouse
embryo including extra-embryonic tissue.
The red region marks embryonic stem cells
in the extra-embryonic yolk sac. Embryonic
stem cells are not normally able to do this,
but when cells are pushed backwards in
development as described in Morgani et al..
Credit: Sophie Morgani, University of
Copenhagen. 

Embryonic stem cells can make all kinds of adult cells in the human body such as muscle, blood or brain cells. However, these embryonic stem cells are created at a point when the embryo has already lost the ability to make extra-embryonic tissue such as placenta and yolk sac. Extra-embryonic tissues are formed at the very earliest stage of development right after fertilization and are essential for the growth of the embryo and its implantation in the womb.

A team of scientists at the Danish Stem Cell Center, DanStem, at the University of Copenhagen have shown that it is possible to rewind the developmental state of embryonic stem cells. By maintaining mouse embryonic stem cells under certain conditions, they found that cells appear to regress and resemble extremely early embryo cells that can form any kind of cell including placenta and yolk sac cells.

"It was a very exciting moment when we tested the theory," says Professor Josh Brickman from DanStem.

"We found that not only can we make adult cells but also placenta, in fact we got precursors of placenta, yolk sac as well as embryo from just one cell."

"This new discovery is crucial for the basic understanding of the nature of embryonic stem cells and could provide a way to model the development of the organism as a whole, rather than just the embryonic portion. In this way we may gain greater insight into conditions where extra-embryonic development is impaired, as in the case of miscarriages," added Sophie Morgani, PhD student at DanStem and first author of the paper.

LIF protein plays a crucial role

Brickman and colleagues grew their embryonic stem cells in a solution containing LIF, which is a protein known to somehow support embryonic stem cells but also for its role in implantation of the embryo into the uterus. As implantation is stimulated by the cells that will become the placenta, not the embryo, these roles appeared to be contradictory. The DanStem study resolved this contradiction by revealing that LIF helps maintain the cells in their regressed, early stage of development.

"In our study we have been able to see the full picture unifying LIF's functions: What LIF really does, is to support the very early embryo state, where the cells can make both embryonic cells and placenta. This fits with LIFs' role in supporting implantation," Josh Brickman says.

Source: University of Copenhagen 

Contact: Joshua Brickman

Reference:

Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

Sophie M. Morgani, Maurice A. Canham, Jennifer Nichols, Alexei A. Sharov, Rosa Portero Migueles, Minoru S.H. Ko, and Joshua M. Brickman
Cell Reports, 06 June 2013, 10.1016/j.celrep.2013.04.034

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