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3D revolution and tissue repair

Posted in 3D Plotting Scaffolds, Bio Instrumentation in Experimental Life Sciences Research, BioInks, BioPrinting in Regenerative Medicine, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Curation, Genome Biology, Medical Devices R&D and Inventions, Specialized 3D BioPrinters (Cornea, Stem Cells for Regenerative Medicine, tagged 3D printing, cell plasticity, coralyne-induced spatial asymmetry, DNA nanostructures, DNA-nanogold conjugates, electron tomography, electron transport, induced multipotent stem cells (iMS), nanocrystal-based field effect transistors, stem cells, tissue engineering scaffolds, tissue repair on April 11, 2016| Leave a Comment »

3D revolution and tissue repair

Curator: Larry H. Bernstein, MD, FCAP

 

 

Berkeley Lab captures first high-res 3D images of DNA segments

DNA segments are targeted to be building blocks for molecular computer memory and electronic devices, nanoscale drug-delivery systems, and as markers for biological research and imaging disease-relevant proteins

www.kurzweilai.net/berkeley-lab-captures-first-high-res-3d-images-of-dna-segments

http://www.kurzweilai.net/images/Cover-fig-512×461.png

In a Berkeley Lab-led study, flexible double-helix DNA segments (purple, with green DNA models) connected to gold nanoparticles (yellow) are revealed from the 3D density maps reconstructed from individual samples using a Berkeley Lab-developed technique called individual-particle electron tomography (IPET). Projections of the structures are shown in the green background grid. (credit: Berkeley Lab)

An international research team working at the Lawrence Berkeley National Laboratory (Berkeley Lab) has captured the first high-resolution 3D images of double-helix DNA segments attached at either end to gold nanoparticles — which could act as building blocks for molecular computer memory and electronic devices (see World’s smallest electronic diode made from single DNA molecule), nanoscale drug-delivery systems, and as markers for biological research and for imaging disease-relevant proteins.

The researchers connected coiled DNA strands between polygon-shaped gold nanoparticles and then reconstructed 3D images, using a cutting-edge electron microscope technique coupled with a protein-staining process and sophisticated software that provided structural details at the scale of about 2 nanometers.

“We had no idea about what the double-strand DNA would look like between the gold nanoparticles,” said Gang “Gary” Ren, a Berkeley Lab scientist who led the research. “This is the first time for directly visualizing an individual double-strand DNA segment in 3D,” he said.

The results were published in an open-access paper in the March 30 edition of Nature Communications.

The method developed by this team, called individual-particle electron tomography (IPET), had earlier captured the 3-D structure of a single protein that plays a key role in human cholesterol metabolism. By grabbing 2D images of an object from different angles, the technique allows researchers to assemble a 3D image of that object.

The team has also used the technique to uncover the fluctuation of another well-known flexible protein, human immunoglobulin 1, which plays a role in the human immune system.

https://youtu.be/lQrbmg9ry90
Berkeley Lab | 3-D Reconstructions of Double strand DNA and Gold Nanoparticle Structures

For this new study of DNA nanostructures, Ren used an electron-beam study technique called cryo-electron microscopy (cryo-EM) to examine frozen DNA-nanogold samples, and used IPET to reconstruct 3-D images from samples stained with heavy metal salts. The team also used molecular simulation tools to test the natural shape variations (“conformations”) in the samples, and compared these simulated shapes with observations.

First visualization of DNA strand dynamics without distorting x-ray crystallography

Ren explained that the naturally flexible dynamics of samples, like a man waving his arms, cannot be fully detailed by any method that uses an average of many observations.

A popular way to view the nanoscale structural details of delicate biological samples is to form them into crystals and zap them with X-rays, but that destroys their natural shape, especially fir the DNA-nanogold samples in this study, which the scientists say are incredibly challenging to crystallize. Other common research techniques may require a collection of thousands of near-identical objects, viewed with an electron microscope, to compile a single, averaged 3-D structure. But an averaged 3D image may not adequately show the natural shape fluctuations of a given object.

The samples in the latest experiment were formed from individual polygon gold nanostructures, measuring about 5 nanometers across, connected to single DNA-segment strands with 84 base pairs. Base pairs are basic chemical building blocks that give DNA its structure. Each individual DNA segment and gold nanoparticle naturally zipped together with a partner to form the double-stranded DNA segment with a gold particle at either end.

https://youtu.be/RDOpgj62PLU
Berkeley Lab | These views compare the various shape fluctuations obtained from different samples of the same type of double-helix DNA segment (DNA renderings in green, 3D reconstructions in purple) connected to gold nanoparticles (yellow).

The samples were flash-frozen to preserve their structure for study with cryo-EM imaging. The distance between the two gold nanoparticles in individual samples varied from 20 to 30 nanometers, based on different shapes observed in the DNA segments.

Researchers used a cryo-electron microscope at Berkeley Lab’s Molecular Foundry for this study. They collected a series of tilted images of the stained objects, and reconstructed 14 electron-density maps that detailed the structure of individual samples using the IPET technique.

Sub-nanometer images next

Ren said that the next step will be to work to improve the resolution to the sub-nanometer scale.

“Even in this current state we begin to see 3-D structures at 1- to 2-nanometer resolution,” he said. “Through better instrumentation and improved computational algorithms, it would be promising to push the resolution to that visualizing a single DNA helix within an individual protein.”

In future studies, researchers could attempt to improve the imaging resolution for complex structures that incorporate more DNA segments as a sort of “DNA origami,” Ren said. Researchers hope to build and better characterize nanoscale molecular devices using DNA segments that can, for example, store and deliver drugs to targeted areas in the body.

“DNA is easy to program, synthesize and replicate, so it can be used as a special material to quickly self-assemble into nanostructures and to guide the operation of molecular-scale devices,” he said. “Our current study is just a proof of concept for imaging these kinds of molecular devices’ structures.”

The team included researchers at UC Berkeley, the Kavli Energy NanoSciences Institute at Berkeley Lab and UC Berkeley, and Xi’an Jiaotong University in China. This work was supported by the National Science Foundation, DOE Office of Basic Energy Sciences, National Institutes of Health, the National Natural Science Foundation of China, Xi’an Jiaotong University in China, and the Ministry of Science and Technology in China. View more about Gary Ren’s research group here.

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Abstract of Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ~2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.

references:

• Gang Ren et al. Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography. Nature Communications, March 2016 DOI: 10.1038/ncomms11083 (open access)

 

World’s smallest electronic diode made from single DNA molecule

Electronic components 1,000 times smaller than with silicon may be possible
http://www.kurzweilai.net/worlds-smallest-electronic-diode-made-from-single-dna-molecule

http://www.kurzweilai.net/images/DNA-diode.jpg

By inserting a small “coralyne” molecule into DNA, scientists were able to create a single-molecule diode (connected here by two gold electrodes), which can be used as an active element in future nanoscale circuits. The diode circuit symbol is shown on the left. (credit: University of Georgia and Ben-Gurion University)

Nanoscale electronic components can be made from single DNA molecules, as researchers at the University of Georgia and at Ben-Gurion University in Israel have demonstrated, using a single molecule of DNA to create the world’s smallest diode.

DNA double helix with base pairs (credit: National Human Genome Research Institute)

A diode is a component vital to electronic devices that allows current to flow in one direction but prevents its flow in the other direction. The development could help stimulate development of DNA components for molecular electronics.

As noted in an open-access Nature Chemistry paper published this week, the researchers designed a 11-base-pair (bp) DNA molecule and inserted a small molecule named coralyne into the DNA.*

They found, surprisingly, that this caused the current flowing through the DNA to be 15 times stronger for negative voltages than for positive voltages, a necessary feature of a diode.

Electronic elements 1,00o times smaller than current components

“Our discovery can lead to progress in the design and construction of nanoscale electronic elements that are at least 1,000 times smaller than current components,” says the study’s lead author, Bingqian Xu an associate professor in the UGA College of Engineering and an adjunct professor in chemistry and physics.

The research team plans to enhance the performance of the molecular diode and construct additional molecular devices, which may include a transistor (similar to a two-layer diode, but with one additional layer).

A theoretical model developed by Yanantan Dubi of Ben-Gurion University indicated the diode-like behavior of DNA originates from the bias voltage-induced breaking of spatial symmetry inside the DNA molecule after the coralyne is inserted.

The research is supported by the National Science Foundation.

*“We prepared the DNA–coralyne complex by specifically intercalating two coralyne molecules into a custom-designed 11-base-pair (bp) DNA molecule (5′-CGCGAAACGCG-3′) containing three mismatched A–A base pairs at the centre,” according to the authors.

UPDATE April 6, 2016 to clarify the coralyne intercalation (insertion) into the DNA molecule.

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Abstract of Molecular rectifier composed of DNA with high rectification ratio enabled by intercalation

The predictability, diversity and programmability of DNA make it a leading candidate for the design of functional electronic devices that use single molecules, yet its electron transport properties have not been fully elucidated. This is primarily because of a poor understanding of how the structure of DNA determines its electron transport. Here, we demonstrate a DNA-based molecular rectifier constructed by site-specific intercalation of small molecules (coralyne) into a custom-designed 11-base-pair DNA duplex. Measured current–voltage curves of the DNA–coralyne molecular junction show unexpectedly large rectification with a rectification ratio of about 15 at 1.1 V, a counter-intuitive finding considering the seemingly symmetrical molecular structure of the junction. A non-equilibrium Green’s function-based model—parameterized by density functional theory calculations—revealed that the coralyne-induced spatial asymmetry in the electron state distribution caused the observed rectification. This inherent asymmetry leads to changes in the coupling of the molecular HOMO−1 level to the electrodes when an external voltage is applied, resulting in an asymmetric change in transmission.

references:

• Cunlan Guo, Kun Wang, Elinor Zerah-Harush, Joseph Hamill, Bin Wang, Yonatan Dubi, Bingqian Xu. Molecular rectifier composed of DNA with high rectification ratio enabled by intercalation. Nature Chemistry, 2016; DOI: 10.1038/nchem.2480
• Cunlan Guo, Kun Wang, Elinor Zerah-Harush, Joseph Hamill, Bin Wang, Yonatan Dubi, Bingqian Xu. Molecular rectifier composed of DNA with high rectification ratio enabled by intercalation. Nature Chemistry, 2016; Supplementary Information (open access)

related:

• A high-performance single-molecule diode

 

A stem-cell repair system that can regenerate any kind of human tissue …including disease and aging; human trials next year
http://www.kurzweilai.net/a-stem-cell-repair-system-that-can-regenerate-any-kind-of-human-tissue

http://www.kurzweilai.net/images/spinal_disc_regeneration.jpg

UNSW researchers say the therapy has enormous potential for treating spinal disc injury and joint and muscle degeneration and could also speed up recovery following complex surgeries where bones and joints need to integrate with the body (credit: UNSW TV)

A stem cell therapy system capable of regenerating any human tissue damaged by injury, disease, or aging could be available within a few years, say University of New South Wales (UNSW Australia) researchers.

Their new repair system*, similar to the method used by salamanders to regenerate limbs, could be used to repair everything from spinal discs to bone fractures, and could transform current treatment approaches to regenerative medicine.

The UNSW-led research was published this week in the Proceedings of the National Academy of Sciences journal.

Reprogramming bone and fat cells

The system reprograms bone and fat cells into induced multipotent stem cells (iMS), which can regenerate multiple tissue types and has been successfully demonstrated in mice, according to study lead author, haematologist, and UNSW Associate Professor John Pimanda.

“This technique is a significant advance on many of the current unproven stem cell therapies, which have shown little or no objective evidence they contribute directly to new tissue formation,” Pimanda said. “We have taken bone and fat cells, switched off their memory and converted them into stem cells so they can repair different cell types once they are put back inside the body.”

“We are currently assessing whether adult human fat cells reprogrammed into iMS cells can safely repair damaged tissue in mice, with human trials expected to begin in late 2017.”

http://www.kurzweilai.net/images/UNSW-stem-cell-repair.jpg

Advantages over stem-cell types

There are different types of stem cells including embryonic stem (ES) cells, which during embryonic development generate every type of cell in the human body, and adult stem cells, which are tissue-specific, but don’t regenerate multiple tissue types. Embryonic stem cells cannot be used to treat damaged tissues because of their tumor forming capacity. The other problem when generating stem cells is the requirement to use viruses to transform cells into stem cells, which is clinically unacceptable, the researchers note.

Research shows that up to 20% of spinal implants either don’t heal or there is delayed healing. The rates are higher for smokers, older people and patients with diseases such diabetes or kidney disease.

Human trials are planned next year once the safety and effectiveness of the technique using human cells in mice has been demonstrated.

* The technique involves extracting adult human fat cells and treating them with the compound 5-Azacytidine (AZA), along with platelet-derived growth factor-AB (PDGF-AB) for about two days. The cells are then treated with the growth factor alone for a further two-three weeks.

AZA is known to induce cell plasticity, which is crucial for reprogramming cells. The AZA compound relaxes the hard-wiring of the cell, which is expanded by the growth factor, transforming the bone and fat cells into iMS cells. When the stem cells are inserted into the damaged tissue site, they multiply, promoting growth and healing.

The new technique is similar to salamander limb regeneration, which is also dependent on the plasticity of differentiated cells, which can repair multiple tissue types, depending on which body part needs replacing.

Along with confirming that human adult fat cells reprogrammed into iMS stem cells can safely repair damaged tissue in mice, the researchers said further work is required to establish whether iMS cells remain dormant at the sites of transplantation and retain their capacity to proliferate on demand.

https://youtu.be/zAMCBNujzzw

Abstract of PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

references:

• Vashe Chandrakanthan, Avani Yeola, Jair C. Kwan, Rema A. Oliver, Qiao Qiao, …, William Walsh, and John E. Pimanda.  PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells. PNAS, April 4, 2016 DOI: 10.1073/pnas.1518244113

related:

• Scientists time-reverse developed stem cells to make them ‘embryonic’ again
• Could humans ever regenerate limbs?

 

First transistors made entirely of nanocrystal ‘inks’ in simplified process

Transistors and other electronic components to be built into flexible or wearable applications; 3D printing planned
http://www.kurzweilai.net/first-transistors-made-entirely-of-nanocrystal-inks

http://www.kurzweilai.net/images/Flexible-Transistors-.jpg

Because this process works at relatively low temperatures, many transistors can be made on a flexible backing at once. (credit: University of Pennsylvania)

University of Pennsylvania engineers have developed a simplified new approach for making transistors by sequentially depositing their components in the form of liquid nanocrystal “inks.” The new process open the door for transistors and other electronic components to be built into flexible or wearable applications. It also avoids the highly complex current process for creating transistors, which requires high-temperature, high-vacuum equipment. Also, the new lower-temperature process is compatible with a wide array of materials and can be applied to larger areas.

Transistors patterned on plastic backing

The researchers’ nanocrystal-based field effect transistors were patterned onto flexible plastic backings using spin coating, but could eventually be constructed by additive manufacturing systems, like 3D printers.

Published in the journal Science,  the study was lead by Cherie Kagan, the Stephen J. Angello Professor in the School of Engineering and Applied Science, and Ji-Hyuk Choi, then a member of her lab, now a senior researcher at the Korea Institute of Geoscience and Mineral Resources. Researchers at Korea University Korea’s Yonsei University were also involved.

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Kagan’s group developed four nanocrystal inks that comprise the transistor, then deposited them on a flexible backing. (credit: University of Pennsylvania)

The researchers began by dispersing a specific type of nanocrystals in a liquid, creating nanocrystal inks. They developed a library of four of these inks: a conductor (silver), an insulator (aluminum oxide), a semiconductor (cadmium selenide), and a conductor combined with a dopant (a mixture of silver and indium). (“Doping” the semiconductor layer of a transistor with impurities controls whether the device creates a positive or negative charge.)

“These materials are colloids just like the ink in your inkjet printer,” Kagan said, “but you can get all the characteristics that you want and expect from the analogous bulk materials, such as whether they’re conductors, semiconductors or insulators.” Although the electrical properties of several of these nanocrystal inks had been independently verified, they had never been combined into full devices. “Our question was whether you could lay them down on a surface in such a way that they work together to form functional transistors.”

Laying down patterns in layers

Such a process entails layering or mixing them in precise patterns.

First, the conductive silver nanocrystal ink was deposited from liquid on a flexible plastic surface that was treated with a photolithographic mask, then rapidly spun to draw it out in an even layer. The mask was then removed to leave the silver ink in the shape of the transistor’s gate electrode.

The researchers followed that layer by spin-coating a layer of the aluminum oxide nanocrystal-based insulator, then a layer of the cadmium selenide nanocrystal-based semiconductor and finally another masked layer for the indium/silver mixture, which forms the transistor’s source and drain electrodes. Upon heating at relatively low temperatures, the indium dopant diffused from those electrodes into the semiconductor component.

“The trick with working with solution-based materials is making sure that, when you add the second layer, it doesn’t wash off the first, and so on,” Kagan said. “We had to treat the surfaces of the nanocrystals, both when they’re first in solution and after they’re deposited, to make sure they have the right electrical properties and that they stick together in the configuration we want.”

Because this entirely ink-based fabrication process works at lower temperatures than existing vacuum-based methods, the researchers were able to make several transistors on the same flexible plastic backing at the same time.

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The inks’ specialized surface chemistry allowed them to stay in configuration without losing their electrical properties. (credit: University of Pennsylvania)

“Making transistors over larger areas and at lower temperatures have been goals for an emerging class of technologies, when people think of the Internet of things, large area flexible electronics and wearable devices,” Kagan said. “We haven’t developed all of the necessary aspects so they could be printed yet, but because these materials are all solution-based, it demonstrates the promise of this materials class and sets the stage for additive manufacturing.”

Because this entirely ink-based fabrication process works at lower temperatures than existing vacuum-based methods, the researchers were able to make several transistors on the same flexible plastic backing at the same time.

3D-printing transistors for wearables

“This is the first work,” Choi said, “showing that all the components, the metallic, insulating, and semiconducting layers of the transistors, and even the doping of the semiconductor, could be made from nanocrystals.”

“Making transistors over larger areas and at lower temperatures have been goals for an emerging class of technologies, when people think of the Internet of things, large area flexible electronics and wearable devices,” Kagan said. “We haven’t developed all of the necessary aspects so they could be printed yet, but because these materials are all solution-based, it demonstrates the promise of this materials class and sets the stage for additive manufacturing.”

The research was supported by the National Science Foundation, the U.S. Department of Energy, the Office of Naval Research, and the Korea Institute of Geoscience and Mineral Resources funded by the Ministry of Science, ICT, and Future Planning of Korea.

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Abstract of Exploiting the colloidal nanocrystal library to construct electronic devices

Synthetic methods produce libraries of colloidal nanocrystals with tunable physical properties by tailoring the nanocrystal size, shape, and composition. Here, we exploit colloidal nanocrystal diversity and design the materials, interfaces, and processes to construct all-nanocrystal electronic devices using solution-based processes. Metallic silver and semiconducting cadmium selenide nanocrystals are deposited to form high-conductivity and high-mobility thin-film electrodes and channel layers of field-effect transistors. Insulating aluminum oxide nanocrystals are assembled layer by layer with polyelectrolytes to form high–dielectric constant gate insulator layers for low-voltage device operation. Metallic indium nanocrystals are codispersed with silver nanocrystals to integrate an indium supply in the deposited electrodes that serves to passivate and dope the cadmium selenide nanocrystal channel layer. We fabricate all-nanocrystal field-effect transistors on flexible plastics with electron mobilities of 21.7 square centimeters per volt-second.

references:

• Ji-Hyuk Choi, Han Wang, Soong Ju Oh, Taejong Paik, …., Christopher B. Murray, Cherie R. Kagan. Exploiting the colloidal nanocrystal library to construct electronic devices. Science, 08 Apr 2016 DOI: 10.1126/science.aad0371

Best textile manufacturing methods for creating human tissues with stem cells

Bioengineers determine three best processes for engineering tissues needed for organ and tissue repair
http://www.kurzweilai.net/best-textile-manufacturing-methods-for-creating-human-tissues-with-stem-cells

http://www.kurzweilai.net/images/tissue-scaffolds.jpg

All four textile manufacturing processes and corresponding scaffold (structure) types studied exhibited the presence of lipid vacuoles (small red spheres, right column, indicating stem cells undergoing random differentiation), compared to control (left). Electrospun scaffolds (row a) exhibited only a monolayer of lipid vacuoles in a single focal plane, while meltblown, spunbond, and carded scaffolds (rows b, c, d) exhibited vacuoles in multiple planes throughout the fabric thickness. Scale bars: 100 μm (credit: S. A. Tuin et al./Biomedical Materials)

Elizabeth Loboa, dean of the Missouri University College of Engineering, and her team have tested new tissue- engineering methods (based on textile manufacturing) to find ones that are most cost-effective and can be produced in larger quantities.

Tissue engineering is a process that uses novel biomaterials seeded with stem cells to grow and replace missing tissues. When certain types of materials are used, the “scaffolds” that are created to hold stem cells eventually degrade, leaving natural tissue in its place. The new tissues could help patients suffering from wounds caused by diabetes and circulation disorders, patients in need of cartilage or bone repair, and women who have had mastectomies by replacing their breast tissue. The challenge is creating enough of the material on a scale that clinicians need to treat patients.

Comparing textile manufacturing techniques

http://www.kurzweilai.net/images/electrospinning.png

Electrospinning experiment: nanofibers are collected into an ethanol bath and removed at predefined time intervals (credit: J. M. Coburn et al./The Johns Hopkins University/PNAS)

In typical tissue engineering approaches that use fibers as scaffolds, non-woven materials are often bonded together using an electrostatic field. This process, called electrospinning (see Nanoscale scaffolds and stem cells show promise in cartilage repair and Improved artificial blood vessels), creates the scaffolds needed to attach to stem cells.

However, large-scale production with electrospinning is not cost-effective. “Electrospinning produces weak fibers, scaffolds that are not consistent, and pores that are too small,” Loboa said. “The goal of ‘scaling up’ is to produce hundreds of meters of material that look the same, have the same properties, and can be used in clinical settings. So we investigated the processes that create textiles, such as clothing and window furnishings like drapery, to scale up the manufacturing process.”

The group published two papers using three industry-standard, high-throughput manufacturing techniques — meltblowing, spunbonding, and carding — to determine if they would create the materials needed to mimic native tissue.

Meltblowing is a technique during which nonwoven materials are created using a molten polymer to create continuous fibers. Spunbond materials are made much the same way but the fibers are drawn into a web while in a solid state instead of a molten one. Carding involves the separation of fibers through the use of rollers, forming the web needed to hold stem cells in place.

http://www.kurzweilai.net/images/carded-scaffold-fabrication.jpg

Schematic of gilled fiber multifilament spinning and carded scaffold fabrication (credit: Stephen A. Tuin et al./Acta Biomaterialia)

Cost-effective methods

Loboa and her colleagues tested these techniques to create polylactic acid (PLA) scaffolds (a Food and Drug Administration-approved material used as collagen fillers), seeded with human stem cells. They then spent three weeks studying whether the stem cells remained healthy and if they began to differentiate into fat and bone pathways, which is the goal of using stem cells in a clinical setting when new bone and/or new fat tissue is needed at a defect site. Results showed that the three textile manufacturing methods proved as viable if not more so than electrospinning.

“These alternative methods are more cost-effective than electrospinning,” Loboa said. “A small sample of electrospun material could cost between $2 to $5. The cost for the three manufacturing methods is between $.30 to $3.00; these methods proved to be effective and efficient. Next steps include testing how the different scaffolds created in the three methods perform once implanted in animals.”

Researchers at North Carolina State University and the University of North Carolina at Chapel Hill were also involved in the two studies, which were published in Biomedical Materials (open access) and Acta Biomaterialia. The National Science Foundation, the National Institutes of Health, and the Nonwovens Institute provided funding for the studies.

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Abstract of Creating tissues from textiles: scalable nonwoven manufacturing techniques for fabrication of tissue engineering scaffolds

Electrospun nonwovens have been used extensively for tissue engineering applications due to their inherent similarities with respect to fibre size and morphology to that of native extracellular matrix (ECM). However, fabrication of large scaffold constructs is time consuming, may require harsh organic solvents, and often results in mechanical properties inferior to the tissue being treated. In order to translate nonwoven based tissue engineering scaffold strategies to clinical use, a high throughput, repeatable, scalable, and economic manufacturing process is needed. We suggest that nonwoven industry standard high throughput manufacturing techniques (meltblowing, spunbond, and carding) can meet this need. In this study, meltblown, spunbond and carded poly(lactic acid) (PLA) nonwovens were evaluated as tissue engineering scaffolds using human adipose derived stem cells (hASC) and compared to electrospun nonwovens. Scaffolds were seeded with hASC and viability, proliferation, and differentiation were evaluated over the course of 3 weeks. We found that nonwovens manufactured via these industry standard, commercially relevant manufacturing techniques were capable of supporting hASC attachment, proliferation, and both adipogenic and osteogenic differentiation of hASC, making them promising candidates for commercialization and translation of nonwoven scaffold based tissue engineering strategies.

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Abstract of Fabrication of novel high surface area mushroom gilled fibers and their effects on human adipose derived stem cells under pulsatile fluid flow for tissue engineering applications

The fabrication and characterization of novel high surface area hollow gilled fiber tissue engineering scaffolds via industrially relevant, scalable, repeatable, high speed, and economical nonwoven carding technology is described. Scaffolds were validated as tissue engineering scaffolds using human adipose derived stem cells (hASC) exposed to pulsatile fluid flow (PFF). The effects of fiber morphology on the proliferation and viability of hASC, as well as effects of varied magnitudes of shear stress applied via PFF on the expression of the early osteogenic gene marker runt related transcription factor 2 (RUNX2) were evaluated. Gilled fiber scaffolds led to a significant increase in proliferation of hASC after seven days in static culture, and exhibited fewer dead cells compared to pure PLA round fiber controls. Further, hASC-seeded scaffolds exposed to 3 and 6 dyn/cm² resulted in significantly increased mRNA expression of RUNX2 after one hour of PFF in the absence of soluble osteogenic induction factors. This is the first study to describe a method for the fabrication of high surface area gilled fibers and scaffolds. The scalable manufacturing process and potential fabrication across multiple nonwoven and woven platforms makes them promising candidates for a variety of applications that require high surface area fibrous materials.

Statement of Significance

We report here for the first time the successful fabrication of novel high surface area gilled fiber scaffolds for tissue engineering applications. Gilled fibers led to a significant increase in proliferation of human adipose derived stem cells after one week in culture, and a greater number of viable cells compared to round fiber controls. Further, in the absence of osteogenic induction factors, gilled fibers led to significantly increased mRNA expression of an early marker for osteogenesis after exposure to pulsatile fluid flow. This is the first study to describe gilled fiber fabrication and their potential for tissue engineering applications. The repeatable, industrially scalable, and versatile fabrication process makes them promising candidates for a variety of scaffold-based tissue engineering applications.

references:

• S A Tuin, B Pourdeyhimi, E G Loboa. Creating tissues from textiles: scalable nonwoven manufacturing techniques for fabrication of tissue engineering scaffolds. Biomedical Materials, 2016; 11 (1): 015017 DOI: 10.1088/1748-6041/11/1/015017 (open access)
• Stephen A. Tuin, Behnam Pourdeyhimi, Elizabeth G. Loboa. Fabrication of novel high surface area mushroom gilled fibers and their effects on human adipose derived stem cells under pulsatile fluid flow for tissue engineering applications. Acta Biomaterialia, 2016; DOI: 10.1016/j.actbio.2016.03.025

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Brain Biobank

Posted in Human neural progenitor cells, Neurohumoral Transmission, Neurological Diseases, Neuroscience, Organ-on-a-Chip & 3D Printing in Life Sciences, Proteins, Proteomics, Regenerative Biology and Medicine, Signaling, Signaling & Cell Circuits, Stem Cells for Regenerative Medicine, Translational Science, tagged biobank, BioP3 device, cell function correlation, EPR spectroscopy, genetics & genomics, α-synuclein, magnetic resonance, monomeric, Neurological treatments, neuropsychiatry, Neuroscience, NMR spectroscopy, protein structure, regenerative medicine, Spinal Cord Injury, spinal cord repair, stem cells, Translational Research on March 29, 2016| Leave a Comment »

Brain Biobank and studies of disease structure correlates

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Unveiling Psychiatric Diseases

Researchers create neuropsychiatric cellular biobank

By DAVID CAMERON   November 12, 2015   http://hms.harvard.edu/news/unveiling-psychiatric-diseases

Image: iStock/mstroz

Researchers from Harvard Medical School and Massachusetts General Hospital have completed the first stage of an important collaboration aimed at understanding the intricate variables of neuropsychiatric disease—something that currently eludes clinicians and scientists.

The research team, led by Isaac Kohane at HMS and Roy Perlis at Mass General, has created a neuropsychiatric cellular biobank—one of the largest in the world.

It contains induced pluripotent stem cells, or iPSCs, derived from skin cells taken from 100 people with neuropsychiatric diseases such as schizophrenia, bipolar disorder and major depression, and from 50 people without neuropsychiatric illness.

In addition, a detailed profile of each patient, obtained from hours of in-person assessment as well as from electronic medical records, is matched to each cell sample.

As a result, the scientific community can now for the first time access cells representing a broad swath of neuropsychiatric illness. This enables researchers to correlate molecular data with clinical information in areas such as variability of drug reactions between patients. The ultimate goal is to help treat, with greater precision, conditions that often elude effective management.

The cell collection and generation was led by investigators at Mass General, who in collaboration with Kohane and his team are working to characterize the cell lines at a molecular level. The cell repository, funded by the National Institutes of Health, is housed at Rutgers University.

“This biobank, in its current form, is only the beginning,” said Perlis, director of the MGH Psychiatry Center for Experimental Drugs and Diagnostics and HMS associate professor of psychiatry. “By next year we’ll have cells from a total of four hundred patients, with additional clinical detail and additional cell types that we will share with investigators.”

A current major limitation to understanding brain diseases is the inability to access brain biopsies on living patients. As a result, researchers typically study blood cells from patients or examine post-mortem tissue. This is in stark contrast with diseases such as cancer, for which there are many existing repositories of highly characterized cells from patients.

The new biobank offers a way to push beyond this limitation.

 

A Big Step Forward

While the biobank is already a boon to the scientific community, researchers at MGH and the HMS Department of Biomedical Informatics will be adding additional layers of molecular data to all of the cell samples. This information will include whole genome sequencing and transcriptomic and epigenetic profiling of brain cells made from the stem cell lines.

Collaborators in the HMS Department of Neurobiology, led by Michael Greenberg, department chair and Nathan Marsh Pusey Professor of Neurobiology,  will also work to examine characteristics of other types of neurons derived from these stem cells.

“This can potentially alter the entire way we look at and diagnose many neuropsychiatric conditions,” said Perlis.

One example may be to understand how the cellular responses to medication correspond to the patient’s documented responses, comparing in vitro with in vivo. “This would be a big step forward in bringing precision medicine to psychiatry,” Perlis said.

“It’s important to recall that in the field of genomics, we didn’t find interesting connections to disease until we had large enough samples to really investigate these complex conditions,” said Kohane, chair of the HMS Department of Biomedical Informatics.

“Our hypothesis is that here we will require far fewer patients,” he said. “By measuring the molecular functioning of the cells of each patient rather than only their genetic risk, and combining that all that’s known of these people in terms of treatment response and cognitive function, we will discover a great deal of valuable information about these conditions.”

Added Perlis, “In the early days of genetics, there were frequent false positives because we were studying so few people. We’re hoping to avoid the same problem in making cellular models, by ensuring that we have a sufficient number of cell lines to be confident in reporting differences between patient groups.”

The generation of stem cell lines and characterization of patients and brain cell lines is funded jointly by the the National Institute of Mental Health, the National Human Genome Research Institute and a grant from the Centers of Excellence in Genomic Science program.

 

On C.T.E. and Athletes, Science Remains in Its Infancy

by BENEDICT CAREY  MARCH 27, 2016   http://www.nytimes.com/2016/03/28/health/cte-brain-disease-nfl-football-research.html

$16 Million for Brain Research, but $0 from N.F.L.

By KEN BELSON   DEC. 22, 2015    http://www.nytimes.com/2015/12/23/sports/football/grant-of-nearly-16-million-for-cte-researchers.html

http://www.nytimes.com/2014/10/13/science/researchers-replicate-alzheimers-brain-cells-in-a-petri-dish.html

https://static01.nyt.com/images/2014/10/13/us/ALZHEIMERS/ALZHEIMERS-master675.jpg

A three-dimensional human neural cell culture model of Alzheimer’s disease

Se Hoon Choi, Young Hye Kim, Matthias Hebisch, et al.

http://www.nature.com/articles/nature13800.epdf

Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles¹. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau^{2, 3}. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology^{4, 5}. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles^{6, 7, 8, 9, 10, 11}. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

 

 

Figure 2: Robust increases of extracellular amyloid-β deposits in 3D-differentiated hNPCs with FAD mutations.close

a, Thin-layer 3D culture protocol. HC, histochemistry; IF, immunofluorescence; IHC, immunohistochemistry. b, Amyloid-β deposits in 6-week differentiated control and FAD ReN cells in 3D Matrigel (green, GFP; blue, 3D6; scale bar, …

 

Stem Cell-Based Spinal Cord Repair Enables Robust Corticospinal Regeneration

March 29, 2016 by mburatov  

In the March 28th, 2016 issue of the journal Nature Medicine, Mark Tuszynski and his colleagues from the University of California, San Diego, in collaboration with colleagues from Japan and Wisconsin, report that they were able to successfully coax stem cell-derived neurons to regenerate damaged corticospinal tracts in rats. Furthermore, this regeneration produced observable, functional benefits.

What is the “corticospinal tract” you ask? The corticospinal tracts are part of the “pyramidal tracts” that include the corticospinal and corticobulbar tracts. The pyramidal tracts are the main controllers of voluntary movement and connect their nerve fibers eventually to cells that serve voluntary muscles and allow them to contract. We call such nerves “motor nerves,” and the corticospinal nerve tracts are among the most important of the motor nerve tracts.

These neural tracts are collectively called “pyramidal tracts” because they pass through a small area of the brain stem known as the pyramids, which lie on the ventral side of the medulla oblongata. Both pyramidal tracts originate in the forebrain; specifically from the so-called “motor cortex” of the forebrain. The motor cortex lies just in front of the central sulcus of the forebrain. In the motor cortex, lies thousands of “upper motor neurons” that extend their axons down to the brain stem and spinal cord.

In the brain stem, the majority of these corticospinal tracts crossover (or decussate) to the other side of the brain stem and travel down the opposite side of the spinal cord. The corticospinal axons extend all the way down the spinal cord, until they make a connection (synapse) with a “lower motor neuron” that extends its axon to the skeletal muscles that it will direct to contract. The corticobulbar tract contains nerves that conduct nerve impulses from cranial nerves and these help the muscles of the face and neck contract, and are involved in facial expressions, swallowing, chewing, and so on.

Damage to the upper motor neurons as a result of a stroke can rob a person of the ability to move, since the muscles that are attached to the upper motor neurons cannot receive any signals to contract. Likewise, damage to the axonal tracts (also known as nerve fibers) can paralyze a patient and rob them of their ability to move.

The director of this research project, Mark Tuszynski, MD, PhD, professor in the UC San Diego School of Medicine Department of Neurosciences and director of the UC San Diego Translational Neuroscience Institute, said: “The corticospinal projection is the most important motor system in humans. It has not been successfully regenerated before. Many have tried, many have failed – including us, in previous efforts.”

“We humans use corticospinal axons for voluntary movement,” said Tuszynski. “In the absence of regeneration of this system in previous studies, I was doubtful that most therapies taken to humans would improve function. Now that we can regenerate the most important motor system for humans, I think that the potential for translation is more promising.”

 

Novel use of EPR spectroscopy to study in vivo protein structure

http://www.news-medical.net/whitepaper/20160315/Novel-use-of-EPR-spectroscopy-to-study-in-vivo-protein-structure.aspx

α-synuclein

α-synuclein is a protein found abundantly throughout the brain. It is present mainly at the neuron ends where it is thought to play a role in ensuring the supply of synaptic vesicles in presynaptic terminals, which are required for the release of neurotransmitters to relay signals between neurons. It is critical for normal brain function.

However, α-synuclein is also the primary protein component of the cerebral amyloid deposits characteristic of Parkinson’s disease and its precursor is found in the amyloid plaques of Alzheimer’s disease. Although α-synuclein is present in all areas of the brain, these disease-state amyloid plaques only arise in distinct areas.

Alpha-synuclein protein. May play role in Parkinson’s and Alzheimer’s disease.  © molekuul.be / Shutterstock.com

Imaging of isolated samples of α-synuclein in vitro indicate that it does not have the precise 3D folded structure usually associated with proteins. It is therefore classed as an intrinsically disordered protein. However, it was not known whether the protein also lacked a precise structure in vivo.

There have been reports that it can form helical tetramers. Since the 3D structure of a biological protein is usually precisely matched to the specific function it performs, knowing the structure of α-synuclein within a living cell will help elucidate its role and may also improve understanding of the disease states with which it is associated.

If α-synuclein remains disordered in vivo, it may be possible for the protein to achieve different structures, and have different properties, depending on its surroundings.

Techniques for determining protein structure

It has long been known that elucidating the structure of a protein at an atomic level is fundamental for understanding its normal function and behavior. Furthermore, such knowledge can also facilitate the development of targeted drug treatments. Unfortunately, observing the atomic structure of a protein in vivo is not straightforward.

X-ray diffraction is the technique usually adopted for visualizing structures at atomic resolution, but this requires crystals of the molecule to be produced and this cannot be done without separating the molecules of interest from their natural environment. Such processes can modify the protein from its usual state and, particularly with complex structures, such effects are difficult to predict.

The development of nuclear magnetic resonance (NMR) spectroscopy improved the situation by making it possible for molecules to be analyzed under in vivo conditions, i.e. same pH, temperature and ionic concentration.

More recently, increases in the sensitivity of NMR and the use of isotope labelling have enabled determinations of the atomic level structure and dynamics of proteins to be determined within living cells¹. NMR has been used to determine the structure of a bacterial protein within living cells² but it is difficult to achieve sufficient quantities of the required protein within mammalian cells and to keep the cells alive for NMR imaging to be conducted.

Electron paramagnetic resonance (EPR) spectroscopy for determining protein structure

Recently, researchers have managed to overcome these obstacles by using in-cell NMR and electron paramagnetic resonance (EPR) spectroscopy. EPR spectroscopy is a technique that is similar to NMR spectroscopy in that it is based on the measurement and interpretation of the energy differences between excited and relaxed molecular states.

In EPR spectroscopy it is electrons that are excited, whereas in NMR signals are created through the spinning of atomic nuclei. EPR was developed to measure radicals and metal complexes, but has also been utilized to study the dynamic organization of lipids in biological membranes³.

EPR has now been used for the first time in protein structure investigations and has provided atomic-resolution information on the structure of α-synuclein in living mammalians^4,5.

Bacterial forms of the α-synuclein protein labelled with 15N isotopes were introduced into five types of mammalian cell using electroporation. Concentrations of α-synuclein close to those found in vivo were achieved and the 15N isotopes allowed the protein to be clearly defined from other cellular components by NMR. The conformation of the protein was then determined using electron paramagnetic resonance (EPR).

The results showed that within living mammalian cells α-synuclein remains as a disordered and highly dynamic monomer. Different intracellular environments did not induce major conformational changes.

Summary

The novel use of EPR spectroscopy has resolved the mystery surrounding the in vivo conformation of α-synuclein. It showed that α-synuclein maintains its disordered monomeric form under physiological cell conditions. It has been demonstrated for the first time that even in crowded intracellular environments α-synuclein does not form oligomers, showing that intrinsic structural disorder can be sustained within mammalian cells.

References

1. Freedberg DI and Selenko P. Live cell NMR Annu. Rev. Biophys. 2014;43:171–192.
2. Sakakibara D, et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 2009;458:102–105.
3. Yashroy RC. Magnetic resonance studies of dynamic organisation of lipids in chloroplast membranes. Journal of Biosciences 1990;15(4):281.
4. Alderson TA and Bax AD. Parkinson’s Disease. Disorder in the court. Nature 2016; doi:10.1038/nature16871.
5. Theillet FX, et al. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 2016; doi:10.1038/nature16531.

 

Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration

Ken Kadoya, Paul Lu, Kenny Nguyen, Corinne Lee-Kubli, Hiromi Kumamaru, Lin Yao, et al.

Nature Medicine(2016)          http://dx.doi.org:/10.1038/nm.4066

The corticospinal tract (CST) is the most important motor system in humans, yet robust regeneration of this projection after spinal cord injury (SCI) has not been accomplished. In murine models of SCI, we report robust corticospinal axon regeneration, functional synapse formation and improved skilled forelimb function after grafting multipotent neural progenitor cells into sites of SCI. Corticospinal regeneration requires grafts to be driven toward caudalized (spinal cord), rather than rostralized, fates. Fully mature caudalized neural grafts also support corticospinal regeneration. Moreover, corticospinal axons can emerge from neural grafts and regenerate beyond the lesion, a process that is potentially related to the attenuation of the glial scar. Rat corticospinal axons also regenerate into human donor grafts of caudal spinal cord identity. Collectively, these findings indicate that spinal cord ‘replacement’ with homologous neural stem cells enables robust regeneration of the corticospinal projection within and beyond spinal cord lesion sites, achieving a major unmet goal of SCI research and offering new possibilities for clinical translation.

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Blood forming precursors in bone marrow

Posted in Bone marrow derived cells, Clinical & Translational, Hematology, Stem Cells for Regenerative Medicine, tagged hemopoiesis, stem cells on March 15, 2016| Leave a Comment »

Blood forming precursors in bone marrow

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Blood stem cells study could pave the way for new cancer therapy

UNIVERSITY OF EDINBURGH

http://media.eurekalert.org/multimedia_prod/pub/web/110842_web.jpg

This image shows the formation of blood stem cells inside the embryonic vessel called dorsal aorta. In green is shown secreted molecule called NOGGIN, which plays an important role in this process. The University of Edinburgh

People with leukaemia could be helped by new research that sheds light on how the body produces its blood supply.

Scientists are a step closer to creating blood stem cells that could reduce the need for bone marrow transplants in patients with cancer or blood disorders.

Enabling scientists to grow the stem cells artificially from pluripotent stem cells could also lead to the development of personalised blood therapies, researchers say.

Blood stem cells are found in bone marrow and produce all blood cells in the body. These cells – known as haematopoietic stem cells (HSCs) – help to restore blood supply in patients who have been treated for leukaemia.

Researchers used a mouse model to pinpoint exactly how HSCs develop in the womb. They showed for the first time how three key molecules interact together to generate the cells, which are later found in adult bone marrow.

The discovery could help scientists to recreate this process in the lab, in the hope that HSCs could one day be developed for clinical use.

Scientists say this fundamental understanding of early development may also have an impact on other diseases that affect blood formation and supply.

###

The research has been published in Nature Communications.

Professor Alexander Medvinsky, of the University of Edinburgh’s MRC Centre for Regenerative Medicine said: “There is a pressing need to improve treatments for diseases like leukaemia and this type of research brings us a step closer to that milestone. The more we understand about how embryos develop these blood stem cells, the closer we come to being able to make them in the lab.”

http://www.ed.ac.uk/news/2016/stem-cells-100316

Céline Souilhol, Christèle Gonneau, Javier G. Lendinez, Antoniana Batsivari, Stanislav Rybtsov, Heather Wilson, Lucia Morgado-Palacin, David Hills, Samir Taoudi, Jennifer Antonchuk, Suling Zhao, Alexander Medvinsky. Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells. Nature Communications, 2016; 7: 10784 DOI: 10.1038/ncomms10784

During embryonic development, adult haematopoietic stem cells (HSCs) emerge preferentially in the ventral domain of the aorta in the aorta–gonad–mesonephros (AGM) region. Several signalling pathways such as Notch, Wnt, Shh and RA are implicated in this process, yet how these interact to regulate the emergence of HSCs has not previously been described in mammals. Using a combination of ex vivo and in vivo approaches, we report here that stage-specific reciprocal dorso–ventral inductive interactions and lateral input from the urogenital ridges are required to drive HSC development in the aorta. Our study strongly suggests that these inductive interactions in the AGM region are mediated by the interplay between spatially polarized signalling pathways. Specifically, Shh produced in the dorsal region of the AGM, stem cell factor in the ventral and lateral regions, and BMP inhibitory signals in the ventral tissue are integral parts of the regulatory system involved in the development of HSCs.

Haematopoietic stem cells (HSCs) lie at the foundation of the adult haematopoietic system, and give rise to cells of all blood lineages throughout the lifespan of an organism. An important property of adult (definitive) haematopoietic stem cells (dHSCs) is that they are capable of long-term reconstitution of the haematopoietic system upon transplantation into irradiated recipients. In the mouse, such cells develop by embryonic stages E10–E11 in the aorta–gonad–mesonephros (AGM) region^{1, 2, 3, 4}. An ex vivo approach showed that the AGM region has a robust autonomous capacity to generate dHSCs¹. The AGM region comprises the dorsal aorta flanked on both sides by the urogenital ridges (UGRs), which contain embryonic rudiments of kidney and mesonephros. HSCs develop in a polarized manner, predominantly in the ventral floor of the dorsal aorta (AoV), more rarely in the dorsal domain of the dorsal aorta (AoD), and are absent in the UGRs^{2, 5, 6, 7}. Localization of dHSCs to the AoV in mouse and human embryos was shown by long-term reconstitution experiments^{5, 6}.

Abundant evidence indicates that during development, a specialized embryonic endothelial compartment known as haematogenic (or haemogenic) endothelium gives rise to haematopoietic stem and progenitors cells^{7, 8, 9, 10}. The haematopoietic programme in various vertebrate models is executed predominantly in the AoV, and is recognized by the expression of essential haematopoietic transcription factors, for example, Runx1 and cKit, and the appearance of clusters of haematopoietic cells budding from the endothelium of the dorsal aorta^{6, 8, 9, 11, 12, 13, 14}.

It is broadly accepted that HSCs develop from the haematogenic endothelium within intra-aortic clusters. This transition involves several consecutive maturation steps of HSC precursors: pro-HSCspre-HSC type Ipre-HSC type IIdHSC^{15, 16, 17}. All these precursors express endothelial markers, such as vascular-endothelial cadherin (VC) and CD31, and sequentially upregulate haematopoietic surface markers: CD41 (pro-HSCs), CD43 (pre-HSC type I) and finally CD45 (pre-HSC type II). This maturation process occurs in the dorsal aorta between E9 and E11. Specifically, pro-HSCs emerge at E9, pre-HSCs Type I appear at E10 and pre-HSCs type II predominantly at E11. Unlike dHSCs, pre-HSCs cannot reconstitute the adult haematopoietic system by direct transplantation and require prior maturation in an embryonic or neonatal environment^{15, 16, 17, 18,19}.

A number of signalling pathways (Notch, Wnt, retinoic acid, interleukin-3 and inflammatory) have been implicated in HSC development; however, a coherent picture is yet to be elucidated^{15, 17, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31}. HSC precursors (pro-HSCs, pre-HSCs type I and pre-HSCs type II) express cKit¹⁷ from early developmental stages. A recent study has shown that the cKit ligand, known as stem cell factor (SCF), is a key regulator driving maturation of these HSC precursors into dHSCs in the AGM region¹⁷, which is in agreement with the marked decline of HSC activity in SCF mutant mice^{32, 33}. In the adult, SCF is critically important for HSC maintenance in the bone marrow niche, mainly in the endothelial compartment³². Sonic Hedgehog (Shh) and bone morphogenetic protein 4 (BMP4) pathways are also important mediators; in zebrafish, these two morphogenes are involved in arterial specification and haematopoietic patterning, respectively^34,35. In the mouse, subaortic BMP4 and Shh/Indian Hedgehog derived from gut were also proposed to be responsible for HSC development^{36, 37}.

During development, interactions between spatially segregated compartments are essential for tissue patterning and specification, and are often mediated by gradients of secreted molecules^{38,39, 40}. Molecules secreted by distant tissues, such as somites, can influence HSC development in the AGM region^{41, 42, 43, 44, 45}. Developing HSCs are embedded in the complex AGM microenvironment, suggesting that HSC development may require signals derived from different compartments of the AGM region. We sought to test this hypothesis. However, the analysis of HSC development in vivo is significantly hampered by low accessibility of embryos developing in utero, fast maturation of dHSCs, lack of uniquely specific markers for HSC precursors and their low numbers in the AGM region. Therefore, we employed here a robust ex vivo culture system that models HSC development in the embryo in combination with functional HSC analysis using in vivolong-term reconstitution assay^{15, 16, 17}. Specifically, to study interactions between AGM subregions, we took advantage of the in vitro reaggregation system that enables close juxtaposition of cell types¹⁵.

We show that interactions between three compartments of the AGM, the AoV, the AoD and the UGRs, are necessary for efficient generation of dHSCs. First, we show that dHSC activity in the isolated E10.5 AoV is limited but can be significantly enhanced by co-culture with the AoD, and that this is mediated at least partly by Shh, secreted dorsally in vivo. Second, while HSC activity in isolated E11.5 AoD is limited, co-culture with a competent AoV microenvironment activates dHSC generation in the AoD. We found that this effect is mediated by SCF, which is secreted abundantly by the AoV stroma in vivo as shown here. Third, we show that downregulation of BMP4 signalling by BMP antagonist Noggin, which is present at high levels in the AoV and especially in intra-aortic clusters as revealed here by in vivo observations, is required for HSC development. Fourth, UGRs, which express high levels of SCF, also enhance HSC development in the dorsal aorta.

Our results based on in vivo observations and ex vivo modelling strongly suggest that juxtaposed, anatomically distinct domains within the AGM region create a complex landscape of interactive signals that underpins HSC development.

Pre-HSCs localize preferentially to the AoV

As dHSCs mature from pre-HSCs, we investigated whether the emergence of dHSC predominantly in the AoV⁶ is a result of asymmetric (ventralized) distribution of pre-HSCs. Dorsal aortae were separated from UGRs and bisected into AoV and AoD (including notochord) as described previously⁶ (Supplementary Fig. 1a). The different domains were then directly transplanted into irradiated mice to detect dHSCs. We first confirmed our previous observation that at E11.5 dHSCs appear almost exclusively in the AoV, although some dHSCs were in the AoD and engrafted few recipients at high level (Supplementary Fig. 1b). Limiting dilution analysis showed that dHSCs are approximately four times more frequent in the AoV compared with AoD. UGRs did not contain HSCs in line with previous reports^{2, 6}.

We then investigated the spatial distribution of pre-HSCs type I and pre-HSCs type II in E10.5–E11.5 embryos using the OP9 co-culture system supplemented with Il3+SCF+Flt3 (termed 3GF), which allows pre-HSCs (which do not engraft by direct transplantation) to mature into dHSC that become detectable by long-term repopulation assay as described previously¹⁶. Doses of transplanted cells (expressed in embryo equivalents, e.e.) were chosen based on the requirements of individual experiments (explained in Methods section). In these experiments (Fig. 1), the dose injected was high (1–2 e.e.) to detect potentially low dHSC numbers in AoD and UGRs.

(a) E10.5 AoV, AoD and UGRs were co-aggregated with OP9 and cultured for 5 days, and the formation of dHSCs was then tested by transplantation into irradiated mice (2 e.e. per recipient; AoV: six independent experiments; AoD: four independent experiments; UGRs: two independent experiments). Dashed line indicates the cutoff for high-level engraftment (>70% donor chimaerism). (b) E11.5 aortas and UGRs were transplanted after reaggregate culture (Ao: 0.2 e.e. per recipient and UGRs: 1 e.e. per recipient; two independent experiments). (c,d) Pre-HSCs type I (VC⁺CD45⁻) (c) or type II (VC⁺CD45⁺) (d) sorted from E11.5 AoV and AoD were co-aggregated with OP9 cells and transplanted after culture (1 e.e. per recipient; two independent experiments). (a–d) Levels of engraftment are plotted, and number of repopulated versus total number of transplanted mice are shown in brackets. Number of embryo equivalents (ee) injected in each experiment are indicated on the graphs. (*P<0.05; ***P<0.005; Mann–Whitney U-test). In all these experiments, tissues were cultured with three growth factors (Flt3I, Il3 and SCF). AGM, aorta–gonad–mesonephros region; Ao, dorsal Aorta; AoV, ventral domain of the dorsal aorta; AoD, dorsal domain of the dorsal aorta; UGRs, urogenital ridges.

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We have shown previously that E10.5 AGM region mainly contains type I pre-HSCs, whereas at E11.5, type I and type II pre-HSCs co-exist¹⁶. Dissected E10.5 AGM regions co-cultured with OP9 in 3GF for 5 days were transplanted into adult irradiated recipients. Out of 21 recipients that received cultured AoV, 20 showed high levels (>70%) of donor-derived long-term haematopoietic chimerism (Fig. 1a). In contrast, only 7 out of 16 recipients of cultured AoD were repopulated at high levels (>70%), while the remaining recipients showed lower or no repopulation (7 and 2, respectively). Cultured UGRs did not produce dHSCs (Fig. 1a). Thus, we conclude that the E10.5 AoD does contain pre-HSCs but at significantly lower numbers than the AoV.

We then investigated whether pre-HSCs localization changes in E11.5 embryos and found that pre-HSCs were still exclusively localized to the dorsal aorta; UGRs carefully separated from the lateral mesenchyme adjacent to the dorsal aorta did not give any repopulation after culture (Fig. 1b). To establish the location of pre-HSCs within the E11.5 dorsal aorta, cell populations enriched for pre-HSCs type I (VC⁺CD45^–) and pre-HSCs type II (VC⁺CD45⁺) were sorted from AoV and AoD, and co-cultured with OP9 stromal cells in the presence of 3GF as described previously¹⁶. We again were able to detect pre-HSC activity in AoD although at lower levels than in AoV. After maturation ex vivo, pre-HSCs type I from AoV and AoD repopulated 7 of 11 and 2 of 8 recipients, respectively (Fig. 1c). Similarly, cultured pre-HSCs type II from AoV and AoD repopulated 11 out of 12 and 4 out of 10 recipients, respectively (Fig. 1d). In all cases, multilineage engraftment was confirmed (Supplementary Fig. 2). These data show that pre-HSCs are significantly enriched in AoV.

Reciprocal inductive interactions between AoD and AoV

To explore hypothetical interactions between AoD and AoV, we made use of a dissociation–reaggregation system that recapitulates HSC development ex vivo¹⁵. This system allowed us to integrate AGM domains in a three-dimensional tissue-like organoid¹⁵ and study their interactions in HSC development. To track the origin of dHSCs, AoV and AoD from wild-type (WT) and green fluorescent protein (GFP) embryos with constitutive expression of GFP⁴⁶ were co-aggregated (termed AoV//AoD co-aggregates) and cultured for 5 days in the presence of 3GF before transplantation (Fig. 2a). Mice transplanted with AoV//AoD co-aggregates can be reconstituted by dHSCs coming from AoD and AoV. The presence of GFP allowed the individual contributions of AoV and AoD to the total repopulation level within the same mouse to be assessed (Fig. 2b,c). This is presented in two separate columns in the graph. Namely, while columns 1 and 3 represent the same recipient mice, the former shows exclusively the contribution of the AoD and the latter shows exclusively the contribution of the AoV into each recipient. To assess the influence of AoD and AoV interaction on HSC development, the repopulation by co-aggregated AoD (column 1) or AoV (column 3) can then be compared with repopulation by independently cultured AoD (column 2) or AoV (column 4). All experiments included reciprocal use of WT and GFP tissues in AoV//AoD co-aggregates, and we observed no difference in repopulation properties between WT and GFP embryos. Homotypic AoV//AoV and AoD//AoD co-aggregates were always used as controls. Note that in these experiments, only 0.2 e.e. were injected per recipient, to ensure that the repopulation levels were not saturated and to allow any inductive effects to be revealed.

(a) Experimental design: the ventral domain (AoV) and the dorsal domain (AoD) of the aorta, and the urogenital ridges (UGRs) from wild-type (WT) and GFP+ embryos were subdissected, and chimeric reaggregates from tissues of these two genotypes were generated. Left column: to test interactions between AoV and AoD, chimeric AoV//AoD reaggregates were generated and transplanted into irradiated recipients after 4–5 days of culture (b,c). Right column: to test interactions between Ao and UGRs, chimeric Ao//UGR reaggregates were generated and transplanted into irradiated recipients after 4–5 days culture (d). GFP+ and/or GFP− donor-derived long-term repopulation allowed us to conclude whether dHSCs originated from AoV, AoD or UGRs. Accordingly, the tissue of origin of donor dHSCs is indicated below each graph. (b) E10.5 aortas from WT and GFP embryos were used to generate chimeric reaggregates as depicted schematically above plots. The reciprocal combination of WT and GFP tissues was used to generate AoV//AoD reaggregates. The tissue source of dHSCs is shown separately in the leftmost (AoD) and rightmost (AoV) columns as indicated below the plot (0.2 e.e. per recipient; two independent experiments). (c) E11.5 aortas from WT and GFP embryos were used to generate chimeric reaggregates. The tissue source of dHSCs is shown separately in the leftmost (AoD) and rightmost (AoV) columns as indicated below the plot (0.2 e.e. per recipient; two independent experiments). (d) E11.5 aortas (Ao) and UGRs from WT and GFP embryos were used to generate Ao//UGR chimeric reaggregates. As depicted schematically above the plot, the reciprocal combination of WT and GFP tissues was used to generate Ao//UGR reaggregates. The tissue source of dHSCs is shown separately in left (Ao) and right (UGRs) columns as indicated below the plot (0.01 e.e. per recipient; six independent experiments). (e) Reaggregation of WT Ao with UGRs generate more dHSCs than Ao alone (0.05 e.e. per recipient; two independent experiments). (b–e) In all these experiments, tissues were cultured with three growth factors.

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Using this approach, we found that the E10.5 AoV generates more dHSCs when combined with AoD than on its own (Fig. 2b, compare two rightmost columns). One day later, E11.5 AoD had no positive influence on dHSC generation by AoV (Fig. 2c, compare two rightmost columns). Conversely, the E11.5 AoD produced more HSCs when reaggregated with the AoV than on its own (Fig. 2c, compare two leftmost columns). This inductive effect of AoV on AoD was not observed at E10.5 (Fig. 2b, compare two leftmost columns). These ex vivo modelling experiments revealed reciprocal stage-specific effects of AoV and AoD on HSC development, which could be explained by the differential release of factors by the two regions and/or by differences in the competency of the target cells to respond to signals.

UGRs enhance HSC development in the dorsal aorta  

SCF expression is involved in polarized HSC development

Figure 3: Involvement of polarized stem cell factor in HSC development.

(a) qRT–PCR on fresh AoV, AoD and UGRs at E10.5 and E11.5 showed high expression levels of stem cell factor (SCF) in AoV and UGRs, compared with AoD (data are mean±s.e.m; *P<0.05, **P<0.01, t-test; three independent experiments). No significant difference was observed between E10.5 and E11.5 expression level in any of the tissues. (b) Expression of SCF-GFP and CD31 determined by immunostaining on thick section (300 μm) of SCF-GFP-positive E10.5 AGM region and on SCF-GFP-negative control. Bars, 50 μm. (c) Expression of SCF in sorted populations from fresh E10.5–E11.5 AoV (V) and AoD (D) determined by qRT–PCR. Endo, endothelial population (VC⁺CD45⁻CD43⁻); type I, pre-HSCs type I (VC⁺CD45⁻CD43⁺); type II, pre-HSCs type II (VC⁺CD45⁺); stroma, stromal population (VC⁻CD45⁻CD43⁻). (*P<0.05, t-test; five independent experiments). (d) E10.5 AoD were cultured as reaggregates in the presence of Il3 and Flt3L with or without SCF and human SCF antagonist (SCF-Rh). (0.5 e.e. per recipient; three independent experiments). (e,f) E11.5 AoD (two independent experiments) (e) and E10.5 AoV (two independent experiments) (f) were cultured as explants with or without SCF (no other cytokines); (0.2 e.e. per recipient).

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Shh signalling enhances dHSC generation

 

Figure 4: Sonic Hedgehog is a positive modulator of pre-HSC type I.

(a) Expression level of Sonic Hedgehog (Shh) in E10.5 and E11.5 AGM region determined by qRT–PCR. (data are mean±s.e.m; *P<0.05, t-test; E10.5: three independent experiments and E11.5: two independent experiments). (b) Patched1 and Gli1 expression in endothelial cells (endo: VC⁺CD45⁻CD43⁻), pre-HSCs type I (I: VC⁺CD45⁻CD43⁺) and type II (II: VC⁺CD45⁺) sorted from E11.5 AoV and AoD (two independent experiments). (c) E10.5 AoV and AoD explants were cultured in presence of Shh recombinant protein before transplantation (AoV: 0.1 e.e. per recipient; two independent experiments and AoD: 0.2 e.e. per recipient; three independent experiments). (d) E10.5 AoV and doxycyline-inducible OP9-Shh were co-aggregated and cultured in presence or absence of doxycycline and/or Hedgehog (Hh) antagonist (200 nM) before transplantation (0.2 e.e. per recipient; two independent experiments). (e) 10.5 AoV and AoD co-aggregated with OP9 were cultured in presence of three growth factors with Hh antagonist before transplantation; (0.2 e.e. per recipient; two independent experiments). (f) E11.5 AoV explants were cultured in presence of Shh recombinant protein before transplantation; (0.2 e.e. per recipient; two independent experiments). (g): E11.5 AGM reaggregates were cultured in presence of Hh antagonist before transplantation; (0.1 e.e. per recipient; two independent experiments). (c,d,f,g) In all these experiments, tissues were cultured without cytokines. Hh anta, Hh antagonist; Dox, doxycycline.

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BMP signalling is downregulated in the dHSC lineage

Figure 5: Bone morphogenetic protein signalling is downregulated in dHSC lineage.

(a) Expression of bone morphogenetic protein 4 (BMP4) at E10.5 determined by qRT–PCR; (data are mean±s.e.m.; *P<0.05, t-test; three independent experiments). (b) Expression of BMP4 in the E10.5 AGM region determined by immunostaining on frozen sections. Bars, 50 μm. Zoomed image shows the subendothelial localization of BMP4 (arrowheads). Bars, 10 μm. (c) Expression of phosphorylated-Smad (P-Smad) in the E10.5 AGM region determined by immunostaining on frozen sections. Bars, 50 μm. (d) Id genes expression in endothelial cells, pre-HSCs type I and type II directly isolated from E10.5 and E11.5 AoV determined by qRT–PCR. Endo, endothelial population (VC⁺CD45⁻CD43⁻); type I, pre-HSCs type I (VC⁺CD45⁻CD43⁺); type II, pre-HSCs type II (VC⁺CD45⁺); stroma, stromal population (VC⁻CD45⁻CD43⁻). (Data are mean±s.e.m.; *P<0.05, **P<0.01; t-test; five independent experiments). (e–g) Expression of P-Smad, CD31 and CD45 in the endothelium and haematopoietic clusters of E10.5 dorsal aorta. White arrowheads indicate cells with pre-HSC type II phenotype (CD31⁺CD45⁺); green arrows show (CD31⁺CD45^−/low) cells budding out of the dorsal aorta and expressing P-Smad; asterisks indicate CD31⁺CD45⁻ cells expressing P-Smad within the endothelium. Bars, 10 μm. A positive control showing P-Smad staining in the dorsal part of the neural tube can be found in h.

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Figure 6: Haematopoietic clusters are exposed to low concentration of BMP4 and high levels of Noggin.

(a) Expression of BMP antagonists at E10.5 determined by qRT–PCR (data are mean±s.e.m.; *P<0.05,***P<0.005; t-test; three independent experiments). (b) Expression of Noggin in the E10.5 AGM region determined by immunostaining on frozen sections. Note the expression of Noggin in the notochord (Nt) as expected. Bar, 50 μm. (c) Expression of Noggin and BMP4 in intra-aortic clusters characterized by cKit and CD31 expression. Note that BMP4 is mainly expressed underneath the dorsal aorta (arrowheads), while Noggin is expressed in the cluster (arrows). Bars, 10 μm. (d) Expression of Noggin in isolated populations from E10.5 and E11.5 AoV (V) and AoD (D) determined by qRT–PCR. (*P<0.05, t-test; five independent experiments). (e) Model showing downregulation of BMP activity in dHSC lineage. BMP4 is mainly expressed in the ventral mesenchyme, while Noggin is found in haematopoeitic clusters. Accordingly, BMP activity, assessed by the phosphorylation of Smad1,5 and 8 (P-Smad), is high in mesenchymal cells underneath the aortic endothelium and in some endothelial cells (CD31⁺CD45⁻) of the aortic endothelium and decreases in the haematopoeitic clusters. While some pre-HSC type I cells (CD31⁺CD45^−/low) exhibit BMP signalling at a low level, acquisition of CD45 (shown in red) is accompanied by a complete loss of BMP activity. EC, endothelial cells; MC, mesenchymal cells; I, pre-HSC type I; II, pre-HSC type II.

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BMP signalling inhibits HSC development

http://www.nature.com/ncomms/2016/160308/ncomms10784/images_article/ncomms10784-f7.jpg

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Interactions between SCF, Shh and BMP signalling pathways

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We have shown previously that during murine embryo development definitive HSCs emerge predominantly in the ventral domain of the dorsal aorta (AoV)⁶. This spatially polarized production of HSCs might be explained by different origins of dorsal and ventral endothelium and/or by asymmetric production of key factors involved in HSC development^{37, 52, 53} and we reasoned that directional inductive interactions between AGM compartments could be involved. Great insight into inductive interactions in various organs has previously been obtained through in vitro modelling³⁹. Here we modelled interactions between AGM domains in a co-culture system, which supports HSC development¹⁵. Using this ex vivo system, we demonstrate that at early stages (E10.5) HSC maturation in the AoV region is enhanced by the presence of the AoD. One day later (E11.5), the AoV microenvironment is able to induce HSC development in the AoD, previously thought to be mostly devoid of HSC activity⁶. We also found that UGRs can enhance HSC production from the dorsal aorta, but cannot generate dHSCs themselves, even under influence of the dorsal aorta. Thus, our data strongly suggest that reciprocal stage-specific inductive AoD//AoV interactions and involvement of UGRs are required for execution of the robust development of HSCs in vivo.

Our data indicate that previously established dorso–ventrally polarized HSC development⁶ is defined by two main factors. First, our current data show that although the AoD contains pre-HSCs (both type I and type II), their numbers are lower than in AoV, in line with lower intra-aortic cluster formation previously described in mouse AoD^{6, 13}. Second, as shown here, dHSCs can be induced in the AoD by the AoV, and therefore the dHSCs deficiency in AoD cannot be explained solely by asymmetric pre-HSC distribution, but may also be influenced by differences in the microenvironment.

To study this, we focused on SCF, Shh and BMP4, whose expressions are dorso–ventrally polarized in the AGM region^{36, 47, 49} (and current data). We found that SCF is an inductive signal that is expressed at high levels in the AoV and UGRs, and can stimulate HSC development in isolated AoD, a region which had previously been considered to be mostly devoid of HSC activity. This is in agreement with a key role of SCF in HSC maturation¹⁷. We found that the aortic endothelial compartment expresses high levels of SCF, suggesting its important role in HSC development comparable to the bone marrow microenvironment of adult HSCs³². Importantly, we found that the pre-HSC type I population expresses SCF suggesting a positive-autocrine loop, which could promote HSC development.

Shh signalling in zebrafish is required for aortic angioblast migration and subsequent arterial specification of the dorsal aorta^{34, 54}. We found that in mouse Shh stimulates and a Hh antagonist inhibits the development of HSCs at E10.5 but not at E11.5, in keeping with a previous study³⁷. The induction of dHSCs in AoV by AoD is also limited to the E10.5 stage. Since Shh is secreted by the notochord (which is included in AoD-dissected tissue), this stage specificity is likely defined by the predominant presence of pre-HSCs type I at E10.5, which express higher levels of Shh signalling components (Ptch1 and Gli1) compared with pre-HSCs type II. By E11.5, the pre-HSC population is mainly represented by type II cells¹⁵. Stage-specific loss of sensitivity to Hh signalling was also described in the developing neural tube⁵⁵. Notably, the poor ability of AoD to develop HSCs despite abundant presence of Shh can also be explained by lower levels of Ptch1 and Gli1 detected in AoD- compared with AoV-derived pre-HSC type I. Our ex vivo modelling data indicate that AoD-derived Shh is an active inducer of HSC development in the AGM region. This conclusion does not exclude the possibility that Shh secreted by the gut could also reach the dorsal aorta³⁷, although by E10.5 these sites are separated by an extended mesentery.

BMP4 signalling is a key factor involved during differentiation of ventral mesoderm and its further specification into haematopoietic cells. In zebrafish, BMP signalling is clearly required during the patterning of the dorsal aorta and for the emergence of dHSCs in the ventral wall³⁴. Its role in mouse is less clear due to the early lethality of BMP mutants⁵⁶. Several lines of evidence point to BMP4 as a good candidate regulating HSC development. Indeed, BMP4 is highly expressed in the ventral mesenchyme underneath the dorsal aorta^{34, 36, 49}; some reports suggested its role in controlling dHSC emergence^{36, 57, 58}. However, the in vitro systems used likely assayed the maintenance of dHSCs, rather than their maturation. It was also reported that BMP4 signalling can define their differentiation potential⁵⁹. BMP4 is also involved in the regulation of essential haematopoietic transcription factors such as Scl/Gata2/Fli1 and Runx1 (refs 60, 61). Here we analysed BMP signalling activity in the dHSC lineage in the AGM region. We show that in vivo the pre-HSC type I to type II transition is accompanied by a downregulation of BMP targets (Id genes). This correlates with our data demonstrating that BMP activity is downregulated in intra-aortic clusters and the observations of others that Runx1 expression is attenuating in the developing HSC lineage^{60, 62, 63}. How is this decrease of BMP activity achieved in vivo, despite the presence of BMP4 in AoV? It has previously been noted that in amphibian embryos several BMP inhibitors are also expressed in AoV³⁴. Similarly, our analysis of the embryo showed high expression of a number of BMP antagonists as well as inhibitory Smad6 and Smad7 in mouse AoV that may counteract BMP4 action in HSC lineage. Furthermore, we found that in the AGM region BMP4 and Noggin are spatially segregated: Noggin being present in haematopoietic clusters and BMP4 being mainly expressed underneath the aortic endothelium. Therefore, maturing HSCs in clusters are exposed to low BMP4 concentration and high concentration of the BMP antagonist Noggin. Furthermore, our qRT–PCR analysis shows that the pre-HSC type I population expresses Noggin, which possibly creates a very effective shield that protects them from BMP4. Accordingly, our ex vivo analysis strongly suggests that downregulation of BMP signalling is functionally important for HSC development in the embryo. Indeed, forced BMP signalling activation by the addition of BMP4, strongly inhibits HSC development, and conversely the addition of Noggin stimulates HSC development in E10.5–E11.5 AGM cultures. These results are in line with recent observation that deletion of Smad4, a common transducer for BMP4/TGFβ signalling, markedly augments the formation of intra-aortic clusters⁶⁴. Our data do not exclude the possibility that BMP4 is essential for specification of mouse dHSCs at earlier stages, as described in the zebrafish model, where BMP signalling is required for HSC development at stages closer to mouse E8.5 (ref. 34).

Our analysis indicates that all three signalling pathways studied can cooperate for HSC development (Fig. 8c). Notably, the interplay of Shh and BMP pathways is broadly involved in development. For example, counter gradients of polarized Shh and BMP signalling in the developing spinal cord specify neuronal subsets along the dorso–ventral axis⁶⁵, and the dorsal aorta resembles the neural tube with inverse orientation of Shh- and BMP-secreting centres³⁴. However, we detected an antagonistic relationship between Shh and BMP pathways. At the molecular level, Shh can induce Noggin and Smad6 expression, thus inhibiting BMP4 signalling. In turn, BMP4 suppresses and, accordingly, Noggin enhances Shh signalling. Cooperation between Shh and Noggin has been previously described as critically important for developmental specification of somitic, neural and hair follicle cells^{66, 67, 68}. Our in vitro data suggest that the feed-forward loop ShhNoggin/NogginShh is also involved in HSC development in vivo.

We propose a model where the polarized secreted factors form complex fields of gradients in vivo, which define an effector zone for optimal HSC development in the dorsal aorta and lead to the ventrally shifted appearance of dHSCs (Fig. 8c). Of interest, intra-aortic clusters are abundant in ventro–lateral positions⁶⁹, which may reflect the position of this zone. The dissection close to such a zone could lead to accidental inclusion of powerful dHSCs in AoD samples observed here. Furthermore, it is possible that spatial segregation of co-operating and spatial overlap of antagonizing factors may also be important for adjustment of HSC development in vivo. Indeed, although the pool of pre-HSCs in the AGM region markedly expands during E9.5–11.5 (Rybtsov et al., submitted), complete maturation of the HSC pool is limited: while the majority of cells reach the pre-HSC type II stage, only one or two dHSCs are generated by the end of E11. Such controlled dynamics of HSC development may be needed to prevent a burst of active haematopoiesis in the AGM region. How exactly HSC maturation dynamics depend on overlapping concentrations of factors requires further analysis. Although ex vivo modelling is a powerful tool to dissect mechanisms of HSC development in vivo, there will likely be some variation in details. For example, spatial polarization in the developing HSC niche may define kinetics of HSC development in vivo.While we have demonstrated spatial polarization in vivo of the factors driving HSC development in our model system, it is currently unclear whether any factors become expressed in a polarized manner within the reaggregates and as such, whether polarization is also a pre-requisite for HSC maturation. Alternatively, if polarization is not required, the entire reaggregate may replicate the optimal zone for HSC development, resulting in massive generation of dHSCs. The distinction between these two scenarios will require further investigation.

In summary, our ex vivo modelling experiments suggest that HSC development in the embryo involves stage-dependent interactions between dorsal, ventral and lateral domains of the AGM region, mediated at least partly by the interplay of SCF, Shh, BMP4 and Noggin. Further detailed analysis will be required to better understand the complexity of the AGM signalling landscape in which HSC development takes place. Such knowledge may lead to development of novel protocols for the generation of definitive HSCs in vitro for clinical applications.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting

Jie He1, Omar Abdel-Wahab2, Michelle K. Nahas1, Kai Wang1, Raajit K. Rampal3, Andrew M. Intlekofer4, et al.
http://www.bloodjournal.org/content/early/2016/03/10/blood-2015-08-664649March 10, 2016

Key Points

• Novel clinically-available comprehensive genomic profiling of both DNA and RNA in hematologic malignancies.

• Profiling of 3696 clinical hematologic tumors identified somatic alterations that impact diagnosis, prognosis, and therapeutic selection

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded (FFPE), blood and bone marrow samples with high accuracy in a clinically relevant timeframe, which is performed in our CLIA-certified CAP-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs and gene fusions, with similar accuracy to lower-throughput assays which focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically-relevant genomic alterations with therapeutic relevance.

Cohesin Ring Rules Blood Stem Cells, Binds Them to Renewal or Expansion

GEN News    http://www.genengnews.com/gen-news-highlights/cohesin-ring-rules-blood-stem-cells-binds-them-to-renewal-or-expansion/81252512/

A genome-wide RNAi screen was used to assess the effects of 15,000 genes on the balance between self-renewal and differentiation of human hematopoietic stem cells (HSCs). The screen identified candidate genes whose knockdown maintained the HSC phenotype during culture. Such findings could lead to better protocols to grow these cells outside the body, potentially making bone marrow transplants more available to patients suffering blood cancers, or even identifying novel genes to target during the treatment of leukemia (left and right panels). Four genes in particular implicated cohesin, a ring-like protein complex that binds to the DNA in all of our cells, in the control of self-renewal versus differentiation in HSCs. Deficiency of cohesin causes an increase in self-renewal and a decrease in differentiation of HSCs. [Cell Reports]

Best known for its ability to regulate the separation of sister chromatids during cell division, the cohesin protein complex, a ring-shaped structure, has shown that it has other powers, such as the facilitation of DNA repair and the modification of transcription. And now, according to scientists based at Lund University, there is evidence that the cohesin complex controls the growth of blood stem cells. More to the point, the cohesin complex determines whether blood stem cells self-renew or differentiate.

The new finding is significant because it can help scientists improve the expansion of blood stem cells outside the body, thus increasing the supply of blood stem cells to patients suffering leukemia or hereditary blood disorders. Besides making bone marrow transplant material more available, the new finding could point scientists to new points of attack for the treatment of blood cancer, which is a disruption between blood stem cell multiplication and maturation.

The Lund University scientists, led by Jonas Larsson, presented their results March 17 in the journal Cell Reports, in an article entitled “Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs.” The article describes how a genome-wide RNA interference (RNAi) screen was performed in primary human CD34⁺ cells. This screen enabled the scientists to identify candidate genes whose knockdown maintained the HSC phenotype during culture.

“A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, and SMC3) among the top 20 genes from the screen,” wrote the authors. “Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro.”

A similar expansion, the authors added, was observed in vivo following transplantation to immunodeficient mice.

“Transcriptome analysis of cohesin-deficient CD34⁺ cells showed an upregulation of HSC-specific genes,” the authors continued. This finding, the authors asserted, demonstrates that when cohesin is deficient, transcription shifts to a more stem cell–like pattern.

“The research is unique as the study of so many genes alongside one another is unprecedented,” said Dr. Larsson. “In addition, we have used human blood stem cells, which is difficult in itself as it is requires the gathering of a large amount of material.”

Of the 15,000 genes that were tested, the Lund team found around 20 candidates with a strong capacity to affect the balance of growth in the blood stem cells. What was striking was that four of these 20 genes were physically connected through cooperation in a protein complex.

“The discovery showed that this protein complex is crucial and has an overarching function in the growth of the blood stem cells,” emphasized Dr. Larsson.

The cohesin complex acts as a sort of brace that holds different parts of the DNA strand together in the cell. The researchers believe that this allows the cohesin complex to control access to the “on/off switches” in DNA and to change the impulses the blood stem cells receive from various genes, thereby affecting cell division. The blood stem cell either multiplies or matures to become a specialized cell with other tasks.

Independently of the Lund researchers’ discovery, other research in the field of blood cancer has recently identified mutations in exactly the same four genes in patients with various forms of blood cancer.

“This is incredibly exciting! Together with the results from our study, this indicates that the cohesin genes are directly and crucially significant in the development of blood cancer,” exclaimed the study’s lead author, Ph.D. candidate Roman Galeev. “Our findings entail a new understanding of how the expansion of blood stem cells is controlled. Eventually, this can lead to new ways of affecting the process, either to prevent the development of cancer or to expand the stem cells for transplant.”

UNPRECEDENTED PRECISION STUDY IDENTIFIES THE FOUR GENES RESPONSIBLE FOR BLOOD STEM CELL DEVELOPMENT.

Posted on March 23, 2016 by Healthinnovations

Unprecedented precision study identifies the four genes responsible for blood stem cell development.

An important element in getting blood stem cells to multiply outside the body is to understand which of the approximately 20 000 genes in the human body control their growth. Now, a study from researchers at Lund University has studied close to 15 000 of these genes alongside each other; a feat which is unprecendented. The team state that they have succeeded in identifying four key genes which, together, govern the growth and multiplication of the stem cells. The opensource study is now being published in the journal Cell Reports.

An important step on the way is to map which of the thousands of human genes affect the regulation of stem cell growth.  The current study used RNA interference, a technique in which individual genes in the stem cells are selectively neutralized, to successfully identify the genes which affect the regulation of blood stem cells.

Results show that the protein complex, consisting of the four genes plus one further gene, is called cohesin and forms a sort of brace which holds different parts of the DNA strand together in the cell. The lab hypothesize that this allows the cohesin complex to control access to the ‘on/off switches’ in the DNA, and to change the impulses the blood stem cells receive from various genes, thereby affecting cell division.  They conclude the blood stem cell either multiplies or matures to become a specialised cell with other tasks.

The group state that clarifying what regulates the balance between multiplication and maturation of blood stem cells could provide the right keys to expanding them outside the body. The go on to add that, in addition, it would enable the identification of new points of attack for the treatment of blood cancer, which is precisely a disruption of the balance between multiplication and maturation.

Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs

Roman Galeev, Aurélie Baudet, Praveen Kumar, …., Anders Kvist, Jonas Larsson

http://dx.doi.org/10.1016/j.celrep.2016.02.082Article Info

Highlights

• •A genome-wide RNAi screen was performed in primary human CD34⁺ cells
• •Several cohesin genes were identified as modifiers of renewal and differentiation
• •Cohesin-deficient HSCs show enhanced reconstitution capacity in vivo
• •Cohesin deficiency induces immediate HSC-specific transcriptional programs

Summary

To gain insights into the regulatory mechanisms of hematopoietic stem cells (HSCs), we employed a genome-wide RNAi screen in human cord-blood derived cells and identified candidate genes whose knockdown maintained the HSC phenotype during culture. A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, andSMC3) among the top 20 genes from the screen. Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro. A similar expansion was observed in vivo following transplantation to immunodeficient mice. Transcriptome analysis of cohesin-deficient CD34⁺ cells showed an upregulation of HSC-specific genes, demonstrating an immediate shift toward a more stem-cell-like gene expression signature upon cohesin deficiency. Our findings implicate cohesin as a major regulator of HSCs and illustrate the power of global RNAi screens to identify modifiers of cell fate.

Human hematopoiesis is maintained by a small number of hematopoietic stem cells (HSCs) that are capable of generating all blood cell lineages at an extremely rapid pace for the entire lifespan of a human being (Orkin and Zon, 2008). HSCs have been studied extensively during the last four decades and are probably the best functionally characterized adult stem cells. However, despite this, the regulatory mechanisms that govern different cellular fate options in HSCs have remained incompletely defined. In particular, it has been challenging to understand the molecular basis of the inherent ability of HSCs to self-renew and preserve their undifferentiated state, which has hampered efforts to expand HSCs ex vivo for therapeutic benefit (Dahlberg et al., 2011). Ex vivo expansion of HSCs would allow for critical improvements of bone marrow transplantation procedures in treatment of malignant and inherited hematological diseases (Chou et al., 2010). Defining the genetic and molecular basis of self-renewal of HSCs is thus important to enhance current cell-therapy strategies, but it is also essential in order to better understand mechanisms behind dysregulated hematopoiesis that may cause leukemia. Genes and pathways balancing cell-fate options between renewal and differentiation in stem cells are often key players in cancer development (Orkin and Zon, 2008).

Figure 1

Genome-wide RNAi Screen in Primitive Human Hematopoietic Cells Defines Genes and Pathways Associated with Cancer Progression and Cell Proliferation

(A) Overview of the experimental outline for the primary screen. 60 million cord blood-derived CD34⁺ cells were transduced with a pooled lentiviral library containing 75,000 shRNAs across six transduction replicates in total. A fraction of the cells were isolated after 72 hr, and proviral inserts were deep sequenced to determine the initial library distribution. Following 20 days of culture, CD34⁺ cells were magnetically isolated and proviral inserts were sequenced again to determine the changes in distribution for all shRNAs.

(B) Relative distribution of shRNAs following 20 days of in vitro culture, ranked from the most enriched to the most depleted. The y axis shows the average enrichment value across six replicate screens.

(C) Gene ontology analysis for all genes represented by multiple shRNAs in the most enriched (10%) fraction.

(D) KEGG pathway analysis showing strong enrichment for cancer-associated pathways among the top-scoring genes.

See also Figure S1 and Table S1.

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1. A Secondary Screen Targeted to CD34⁺CD38⁻CD90⁺CD45RA⁻Cells Identifies the Cohesin Complex as a Candidate Regulator of HSC Renewal and Differentiation
2. Knockdown of Cohesin Genes Restricts Differentiation and Expands Primitive Cells In Vitro
3. Cohesin-Deficient HSCs Show Delayed Differentiation but Overall Increased Reconstitution Potential following Transplantation to Primary and Secondary Immunodeficient Mice
4. Silencing of Cohesin Genes Results in Immediate Transcriptional Changes with a Shift toward a More HSC-like Gene Expression Signature

We report here on the successful development of a genome-wide RNAi screening approach targeted to primary human hematopoietic stem and progenitor cells to define genes and pathways associated with self-renewal and differentiation. Based on findings from the screen, we implicate the cohesin complex as a crucial regulator of cell-fate decisions influencing self- renewal and differentiation in HSCs both in vitro and in vivo.

These efforts represent a genome-wide RNAi screen targeted to primary human HSPCs. The main limiting factor when performing functional screens in primary human cells is cell number. This obviously becomes even more challenging when rare cell subsets, such as stem and progenitor cells, are studied. Through unique access to cord blood with daily deliveries from several local hospitals, we were able to gather the necessary quantities to perform a screen in enriched primary HSPCs with reasonable coverage (300X).

 

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