Stem Cell Clinic

Here’s the first 3D glimpse of how DNA is packaged up in a single cell – Ars Technica

Enlarge / Intact genome from a mouse embryonic stem cell with 20 chromosomes colored differently

In a first, researchers have worked out a way to unravel and model the tangled, 3D structures of intact mammalian genomes from individual cells.

The new method, published Monday in Nature, could help researchers study how the complex loops, twists, and bunches of a tightly packaged genome influence which bits of the blueprints are actively used by the cells, and when.

In humans, for instance, genome packaging bundles nearly two meters worth of DNA strings into a nucleus about 0.005 millimeters wide. How all that DNA is bundled affects whether important genes are available for decoding by cellular machinery, while others are boxed up and shoved aside until theyre needed. Such carefully orchestrated genetic activity affects everything a cell doesfrom carrying out basic functions, to allowing stem cells to differentiate into any type of cell, to triggering diseases.

Intact genome from a mouse embryonic stem cell with 20 chromosomes colored differently.

Tom Collins, a genetics and molecular scientist at Wellcome Trust, who was not a study author, called the new, more detailed method an exciting step forward. This detail will reveal some of the underlying principles that govern the organization of our genomesfor example, how chromosomes interact or how structure can influence whether genes are switched on or off, he said in a statement. If we can apply this method to cells with abnormal genomes, such as cancer cells, we may be able to better understand what exactly goes wrong to cause disease and how we could develop solutions to correct this.

To pull off the high resolution look at DNA-packaging, researchers at the University of Cambridge and the Wellcome TrustMRC Stem Cell Institute joined colleagues to combine cell images and a molecular technique called Hi-C. First, the researchers took super zoomed-in images of eight mouse embryonic stem cells genomes. Next, researchers turned to Hi-C, an established method to reveal the structure of genome packaging based on DNA sequences that are caught residing near each other. (Hi-C isnt an acronym. Its a riff on 3C, a shorthand for Chromosome Conformation Capture Analysis.)

Basically, in Hi-C, the cells are fixed with formaldehyde so that bits of DNA held together by protein packing tapestay stuck together. Then researchers use enzymes that act like molecular scissors and snip the genome into tiny fragments. This leaves scraps of DNA strings held together by packaging proteins that are themselves held together by the formaldehyde.

Imagine two short pieces of DNA pinched together at the middle by a piece of tape, creating an X shape. Researchers then fuse those dangling DNA fragments together and create a single piece of DNA from the two fragment that were once just physically close together. After stripping away the tape, the researchers can sequence across the fusion points and reveal which bits of DNA were close to each other thanks to packaging.

This is the structure of a mouse embryonic stem cell genome. The blue indicates active genes, while the yellow indicates genes interacting with the membrane.

From the the eight stem cells, processed individually, the researchers captured between 37,000 and 122,000 DNA junctions. This represents just 1.2 to 4.1 percent of the total possible junctions that could have been in the genomes. But, combined with the high resolution images, they captured enough to assemble 3D structures. When the researchers overlaid data indicating which genes were active or dormant onto their 3D structures, that data squared with the genes positions within the messy balls.

"Knowing where all the genes and control elements are at a given moment will help us understand the molecular mechanisms that control and maintain their expression," saidErnest Laue, lead study author and biochemist at Cambridge.

Nature, 2017. DOI: 10.1038/nature21429 (About DOIs).

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Here's the first 3D glimpse of how DNA is packaged up in a single cell - Ars Technica

The craftsmanship of mimicking embryogenesis in a dish – BioNews

The regulatory mechanisms governing organ development are, in general, poorly defined. To recreate the complex processes involved in organ growth and maturation, scientists have started fiddling with three-dimensional (3D) cell culture. The 3D self-organisation cell/tissue culture approach is conceptually quite different from standard tissue engineering, where cells are cultured in a flat 2D environment. The idea behind itis that realistic tissue formation is dictated both by the internal processes of the cells, and their interactions with the surrounding environment, including communication with neighbouring cells. Therefore, in vivo tissue development could be more accurately simulated by placing cells within an optimal 3D microenvironment.

A breakthrough happened in 2011, when a Japanese group from RIKEN Centre for Developmental Biology in Kobe reported that in a 3D culture of mouse embryonic stem cell aggregates, the cells self-organised to form organoid tissues including an eyecup-like structure and a functional frontal part of the pituitary gland (1, 2). Although mice are not men, in biological science a standard path is to be able to reproduce in human systems what was discovered first in mice. Indeed, a couple of years later, a team from Austria grew cerebral organoids with discrete brain regions by placing human pluripotent stem cells in a 3D culture system (3).

The next major game-changer in the field of organoids was a study reporting self-assembly of functional human liver 'buds'. Liver buds are functional units formed at the early stages of organ development (4). This time, the starting point was not 3D aggregates of one pluripotent stem cell line; instead, three different cell types liver cells derived from induced pluripotent stemcells, connective tissue stem cells, and blood vessel cells were mixed in a specific ratio, which led to the self-assembly of a functional liver bud. Such functional organoids, built from several distinct cell types, represent a new generation of organoids. By using advanced genome editing techniques, such as CRISPR, to interfere with a single gene expression or function in each of the participating cell types, we will be able to study the complex cell communication signals that govern organogenesis. We will be able to define which cell type is providing which signalling molecules and what their roles are establishing the different cell types that make up tissue.

The laboratory of Professor Magdalena Zernicka-Goetz from the University of Cambridge used a similar approach to generate structures highly resembling post-implantation mouse embryos (5). They combined a single mouse embryonic stem cell with a small clump of three trophoblast stem cells (stem cells that would form the placenta), and cultured them within Matrigel, a 3D extracellular matrix (ECM) scaffold, in a medium that allowed both cell types co-develop. To examine the cross-talk between the two cells types and the signalling pathways involved, the team used cell lines with specific mutations in one of the two cell types, and monitored how lack of a specific gene and/or signal affected the morphogenesis of an embryo-like structure.

I do not see why we would not be technically able to mimic human embryogenesis in a dish using a similar approach. However, I could see how that can be viewed with a touch of controversy even though, in my mind, there are no ethical issues or controversy at all. Although the embryo-like structures might resemble human embryos in vivo, they would have no potential to develop into a live organism. To those prophets who would see such development as a collapse of ethos and humanity, I want to say that I am pretty confident that in the future women will still be a part of our society. They will still be pregnant and they will still deliver babies. They will not be replaced with incubators nurturing endless copies of some future version of Kim Jong-un, Vladimir Putin or Donald Trump. Pregnancy and fetal development are too complex to be simulated.

The future of this work, as I see it, is not only as a 'powerful platform to dissect physical and molecular mechanisms that mediate critical crosstalk during natural embryogeneisis', as the Cambridge team say (5), but also as a sophisticated model for human embryotoxicity screening, to learn more about embryo development and the causes of infertility. Currently used in vitro testing protocols are based on animal embryos, not human embryos, and therefore lack specificity and predictability. This model has the potential to be used for large-scale throughput screening of the multiple stages of human embryo development. By studying and testing these stages, we can learn about the growth and differentiation processes of the embryo in great detail. Furthermore, we can test in vitro whether potential drug candidates affect any of these early embryo development stages. In such a way the embryotoxic or cancerous potential of an agent could be easily assessed in a human system, and many unexplained infertility cases classified as 'infertility of unknown etiology' could be resolved and appropriately addressed.

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The craftsmanship of mimicking embryogenesis in a dish - BioNews