Aussies pioneer new approach to mend broken hearts

A printer that builds beating 3D hearts in a laboratory could soon be saving the lives of heart attack patients if a pioneering Australian trial proves successful.

The Heart Research Institute has bought a 3D bio-printer to engineer human heart tissue that can be stuck directly to a damaged organ following an attack.

The Australia-first experiment could dramatically alter the cardiac treatment landscape, giving patients whose hearts are wounded by an attack a chance to fully recover and return to normal life.

“If we can make it work we’ll be changing the lives of thousands of Australians and flipping the world of heart attack treatment on its head.” 

- Dr Carmine Gentile, HRI research fellow and world leader in 3D tissue culture.

More than 350,000 Australians will have a heart attack in their lifetime, and while there have been improvements in recovery rates, the event still claims the lives of 24 Australians every day.

The two common treatments, 

• coronary angioplasty and 
• reperfusion therapy, 

offer excellent results for patients who receive them very soon after an attack, but those who miss out often suffer irreversible heart damage.

With an urgent need for new treatments, the HRI investigated the benefits of a bio-printer to print out unique 3D human mini hearts developed by Dr Gentile. These mini hearts, called cardiac spheroids, were built from stem cells to be used as "bio-ink" to print cardiac patches.

The HRI was awarded a $44,000 grant by the Ian Potter Foundation to purchase the bio-printer, which will first be used to print patches for testing on animal models in the laboratory prior to their use in humans.

“The idea is these patches will be grafted on to the heart after a heart attack, promoting muscle regeneration and returning cardiac function to normal,” Dr Gentile says.

The printer works by spreading “bio-ink” made from human cells layer-by-layer onto biocompatible sheet, called hydrogel, to generate living 3D bio-printed heart tissues. These bio-printed heart tissues can be used in the lab to safely test new heart drugs and to discover new molecular targets for future therapies.

Ultimately though, the technology will be used to replace the damaged parts of human hearts following heart failure and heart attack. As Dr Gentile explains, the printed patches would be customized to each patient to ensure the best outcome.

“Each patient’s heart is scanned first to map the damage to make sure the patch is the right size and shape,” he says. “We’ll also isolate stem cells from the patient’s own skin to make personalized bio-ink to build compatible heart tissue.”

The patch moulds to the heart, mimicking the role of the original damaged tissue for the rest of the individual’s life.

While plenty of past studies have highlighted the feasibility of 3D printing to improve heart disease outcomes in humans, nobody has yet proved it. If successful, the HRI trial would be the first.

Professor Gemma Figtree at the Kolling Institute who is a collaborating partner on the research, is optimistic about the outcome.

“Dr Gentile’s cardiac spheroids have already shown very promising results in the laboratory,” she says. “They have been successful in overcoming hurdles faced by approaches that use only single cells. The future looks very exciting indeed.”

The researchers expect the therapy could be available for patients in the next five years, and every effort will be made to get the treatment into hospitals as soon as possible. The process will be costly however, as it is expensive to collect biological material like cells to 3D bio-print a human cardiac patch.

Listen: Dr Gentile speaks with ABC's The Conversation Hour

"Aussies Pioneer New Approach to Mend Broken Hearts"

ORIGINAL: Heart Research Institute Australia

Kate Rubins’ Space Station Science Scrapbook

As a child, Kate Rubins dreamed of being an astronaut and a scientist. During the past four months aboard the International Space Station, that dream came full circle. She became the first person to sequence DNA in space, among other research during her recent mission, adding to her already impressive experience. She holds a doctorate in molecular biology, and previously led a lab of 14 researchers studying viruses, including Ebola.

Here’s a look back at Rubins in her element, conducting research aboard your orbiting laboratory.

Kate inside Destiny, the U.S. Laboratory Module

Destiny houses the Microgravity Science Glovebox (MSG), in which Kate worked on the Heart Cells experiment.

The U.S. national laboratory, called Destiny, is the primary research laboratory for U.S. payloads, supporting a wide range of experiments and studies contributing to health, safety, and quality of life for people all over the world. 

Swabbing for Surface Samples

Microbes that can cause illness could present problems for current and future long duration space missions. 

Understanding what microbe communities thrive in space habitats could help researchers design antimicrobial technology. Here, Kate is sampling various surfaces of the Kibo module for the Microbe-IV investigation.

Culturing Beating Heart Cells in Space

The Heart Cells investigation uses human skin cells that are induced to become stem cells, which can then differentiate into any type of cell.

Researchers forced the stem cells to grow into human heart cells, which Rubins cultured aboard the space station for one month.

Rubins described seeing the heart cells beat for the first time as “pretty amazing. First of all, there’s a few things that have made me gasp out loud up on board the [space] station. Seeing the planet was one of them, but I gotta say, getting these cells in focus and watching heart cells actually beat has been another pretty big one.”

Innovative Applied Research Experiment from Eli Lilly

The Hard to Wet Surfaces investigation from Eli Lilly, and sponsored by the Center for the Advancement of Science in Space (CASIS), looks at liquid-solid interactions and how certain pharmaceuticals dissolve, which may lead to more potent and effective medicines in space and on Earth. 

Rubins set up vials into which she injected buffer solutions and then set up photography to track how tablets dissolved in the solution in microgravity.

Capturing Dragon

Rubins assisted in the capture of the SpaceX Dragon cargo spacecraft in July. The ninth SpaceX resupply mission delivered more than two thousand pounds of science to the space station. 

Biological samples and additional research were returned on the Dragon spacecraft more than a month later. 

Sliding Science Outside the Station

Science doesn’t just happen inside the space station. External Earth and space science hardware platforms are located at various places along the outside of the orbiting laboratory. 

The Japanese Experiment Module airlock can be used to access the JEM Exposed Facility. Rubins installed the JEM ORU Transfer Interface (JOTI) on the JEM airlock sliding table used to install investigations on the exterior of the orbiting laboratory.

Installing Optical Diagnostic Instrument in the MSG

Rubins installed an optical diagnostic instrument in the Microgravity Science Glovebox (MSG) as part of the Selective Optical Diagnostics Instrument (SODI-DCMIX) investigation. Molecules in fluids and gases constantly move and collide. 

When temperature differences cause that movement, called the Soret effect, scientists can track it by measuring changes in the temperature and movement of mass in the absence of gravity. Because the Soret effect occurs in underground oil reservoirs, the results of this investigation could help us better understand such reservoirs.

The Sequencing of DNA in Space

When Rubins’ expedition began, DNA had never been sequenced in space. Within just a few weeks, she and the Biomolecule Sequencer team had sequenced their one billionth “base” – the unit of DNA - aboard the orbiting laboratory. 

The Biomolecule Sequencer investigation seeks to demonstrate that DNA sequencing in microgravity is possible, and adds to the suite of genomics capabilities aboard the space station.

The MinION™ DNA sequencer from Oxford Nanopore Technologies fits in the palm of a hand.
Credits: Oxford Nanopore Technologies

Studying Fluidic Dynamics with SPHERES

The SPHERES-Slosh investigation examines the way liquids move inside containers in a microgravity environment. The phenomena and mechanics associated with such liquid movement are still not well understood and are very different than our common experiences with a cup of coffee on Earth.

Rockets deliver satellites to space using liquid fuels as a power source, and this investigation plans to improve our understanding of how propellants within rockets behave in order to increase the safety and efficiency of future vehicle designs. Rubins conducted a series of SPHERES-Slosh runs during her mission.

Retrieving Science Samples for Their Return to Earth

Precious science samples like blood, urine and saliva are collected from crew members throughout their missions aboard the orbiting laboratory. 

They are stored in the Minus Eighty-Degree Laboratory Freezer for ISS (MELFI) until they are ready to return to Earth aboard a Soyuz or SpaceX Dragon vehicle.

Measuring Gene Expression of Biological Specimens in Space

Rubins ran several WetLab-2 RNA SmartCycler sessions during her mission.

Our WetLab-2 hardware system is bringing to the space station the technology to measure gene expression of biological specimens in space, and to transmit the results to researchers on Earth at the speed of light. 

Studying the First Expandable Habitat Module on the Space Station

The Bigelow Expandable Activity Module (BEAM) is the first expandable habitat to be installed on the space station. It was expanded on May 28, 2016. 

Expandable habitats are designed to take up less room on a spacecraft, but provide greater volume for living and working in space once expanded. Rubins conducted several evaluations inside BEAM, including air and surface sampling.

Better Breathing in Space and Back on Earth

Airway Monitoring, an investigation from ESA (the European Space Agency), uses the U.S. airlock as a hypobaric facility for performing science. Utilizing the U.S. airlock allows unique opportunities for the study of gravity, ambient pressure interactions, and their effect on the human body. 

This investigation studies the occurrence and indicators of airway inflammation in crew members, using ultra-sensitive gas analyzers to evaluate exhaled air. This could not only help in spaceflight diagnostics, but that also hold applications on earth within diagnostics of similar conditions, for example monitoring of asthma.

Hot Science with Cool Flames

Fire behaves differently in space, where buoyant forces are removed. Studying combustion in microgravity can increase scientists’ fundamental understanding of the process, which could lead to improvement of fire detection and suppression systems in space and on Earth. 

Many combustion experiments are performed in the Combustion Integration Rack (CIR) aboard the space station. Rubins replaced two Multi-user Droplet Combustion Apparatus (MDCA) Igniter Tips as part of the CIR igniter replacement operations.

Though Rubins is back on Earth, science aboard the space station continues, and innovative investigations that seek to benefit humans on Earth and further our exploration of the solar system are ongoing. Follow @ISS_Research to keep up with the science happening aboard your orbiting laboratory. 

Make sure to follow us on Tumblr for your regular dose of space: http://nasa.tumblr.com

nasa space spacestation astrokate astronaut research technology

ORIGINAL: NASA

558 notes Nov 3rd, 2016

Watch This Amazing 3D Bioprinter Make Artificial Bones From Scratch

Image credit: Aether

If 3D printing is already impacting manufacturing today, what breakthroughs could bioprinting — or printing any mix of organic and inorganic materials — achieve tomorrow? In a recent video, a basic prototype of the Aether 1 bioprinter is shown printing two bones connected by a tendon using six materials that include synthetic bone, conductive ink, stem cells and graphene oxide.

While bioprinted organs are still a long way off — this video offers a glimpse into that future.

According to Aether, the printer works with a wide range of materials — organic, non-organic and both — and is flexible about where those materials come from (instead of requiring anything proprietary). It can include 10 different materials in one print (way more than your average bioprinter) and is compatible with other tools like laser cutters and CNC routers. The printer’s universal tool mounts can even be used with pencils and paintbrushes—or whatever else — for making designs on canvas or other materials.

Such a range of possibilities begins to shift the limitations from the hardware to the imagination of the user. What really stands out in this video is the final product — fabricated bones seeded with two kinds of stem cells hooked together with tendons, transistors, and conductive wires. And this is only a test. What else will researchers and makers come up with?

Of course, if it’s not already obvious, their creation isn’t ready for implantation in anyone, though it does show off the machine’s versatility in an eye-catching way. And beyond versatility, the real selling point may be its price. Aether hopes to offer their printer for around $9,000 — while other bioprinters can cost up to $200,000. 

For such bold claims — can they deliver? The announcement and video is only the first in a series which, according to Aether, will show off even greater functionality. Similar price reductions in 3D printers (that don’t bioprint) have met challenges commercially. But falling prices are a key trend to keep track of in any digitally driven technology like 3D printing.

And it should be noted, there are other companies wading into bioprinting too.

An early player, Organovo is currently working with researchers to use 3D printed tissues to test drug toxicity, and last year, they announced a partnership with cosmetics giant L’Oréal to test beauty products on3D printed skin samples (and hopefully lessen reliance on animal testing). Meanwhile, BioBots is making a bioprinter about the same size and cost as Aether’s but with more focused use cases, like 3D printing tissues for research purposes.

We’ll have to wait for more substantial news to see just how all this fits together. This summer will see the first wave of Aether machines donated to universities and researchers while presale beta units are distributed to customers. By fall 2016, they hope to launch retail sales.

What would you use it for?

ORIGINAL: Singularity Hub

BY ANDREW O'KEEFE

ON JUL 08, 2016

Creating 3-D tissue and its potential for regeneration

Bioprinting technique may provide potential for tissue repair and regenerative medicine

Researchers are one step closer to embedding vascular networks into thick human tissues, which could result in tissue repair and regeneration — and ultimately even replacement of whole organs.

A team at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School for Engineering and Applied Sciences (SEAS) has invented a method for 3-D bioprinting thick vascularized tissue constructs. The vasculature network enables fluids, nutrients, and cell growth factors to be perfused uniformly throughout the tissue.

The advance was reported Monday in the journal Proceedings of the National Academy of Sciences.

“This latest work extends the capabilities of our multi-material bioprinting platform to thick human tissues, bringing us one step closer to creating architectures for tissue repair and regeneration,” says the study’s senior author, Jennifer A. Lewis, who is a Wyss core faculty member and the Hansjörg Wyss Professor of Biologically Inspired Engineering at SEAS.

Printing Vascular Tissue

Printing vessel vasculature is essential for sustaining functional living tissues. Until now, bioengineers have had difficulty building thick tissues, lacking a method to embed vascular networks. Credit: Lewis Lab/ Wyss Institute at Harvard University

In the study, Lewis and her team showed that their 3-D printed, vascularized tissues could thrive and function as living tissue architectures for upwards of six weeks.

To date, scaling up human tissues built of a variety of cell types has been limited by an inability to embed life-sustaining vascular networks. Building on their earlier work, Lewis and her team have now increased the tissue thickness threshold nearly tenfold, setting the stage for future advances in tissue engineering and repair. The method combines vascular plumbing with living cells and an extracellular matrix, enabling the structures to function as living tissues.

As an example of what can be done with the technology, Lewis’ team printed 1-centimeter-thick tissue containing human bone marrow stem cells surrounded by connective tissue. By pumping bone growth factors through supporting vasculature lined with the same endothelial cells found in human blood vessels, the scientists induced the cells to develop into bone cells over the course of one month, according to the study.

“This research will help to establish the fundamental scientific understanding required for bioprinting of vascularized living tissues,” said Zhijian Pei, National Science Foundation program director for the Directorate for Engineering Division of Civil, Mechanical, and Manufacturing Innovation, which funded the project. “Research such as this enables broader use of 3-D human tissues for drug safety and toxicity screening and, ultimately, for tissue repair and regeneration.”

Lewis’ novel 3-D bioprinting method uses a customizable, printed silicone mold to house the printed tissue structure. Inside this mold, layers of vascular channels made of pluronic (a material that liquefies at refrigerator temperature) and living stem cells are interdigitated like locking fingers. A cellular matrix is poured around this structure, and solidifies. The entire device is then refrigerated until the pluronic turns to liquid and is sucked out by a vacuum. This creates channels through which liquid containing endothelial cells, oxygen, nutrients, and growth factors — basically, simulated blood — can flow.

The bioprinted material can be used to create living tissue cultures as well as to drive directed tissue growth such as differentiating stem cells. To achieve a variety of tissue shapes, thicknesses, and composition, the shape of the printed silicone chip can be customized and the printable cellular material can be tuned to include a wide variety of cell types. In other words, this new method creates a fully controllable, living 3-D tissue environment, researchers say.

“Having the vasculature prefabricated within the tissue allows enhanced cell functionality at the deep core of the tissue, and gives us the ability to modulate those cell functions through the use of perfusable substances such as growth factors,” said David Kolesky, a graduate researcher at the Wyss Institute and SEAS and one of the study’s first authors.

“Jennifer and her team are shifting the paradigm in the field of tissue engineering based on their unique bioprinting approach,” said Wyss Institute Director Donald Ingber. “Their ability to build living 3-D vascularized tissues from the bottom up provides a potential way to form macroscale functional tissue replacements that can be surgically connected to the body’s own blood vessels to provide immediate perfusion of these artificial tissues, and thus, greatly increase their likelihood of survival. This would overcome many of the problems that held back tissue engineering from clinical success in the past.”

Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the vascular biology program at Boston Children’s Hospital, and professor of bioengineering at SEAS. In addition to Lewis and Kolesky, other team members on the new study include co-first authors Kimberly Homan, research associate at the Wyss Institute, and Mark Skylar-Scott, postdoctoral fellow at the Wyss Institute.

The work was supported by the National Science Foundation and the Wyss Institute for Biologically Inspired Engineering at Harvard University.

Adapted from a Wyss Institute press release written by Kat J. McAlpine, Wyss Institute Communications.

ORIGINAL: Harvard Gazzette

March 8, 2016

First almost fully-formed human brain grown in lab, researchers claim

Research team say tiny brain could be used to test drugs and study diseases, but scientific peers urge caution as data on breakthrough kept under wraps

The tiny brain, which resembles that of
a five-week-old foetus, is not conscious.
Photograph: Ohio State University

An almost fully-formed human brain has been grown in a lab for the first time, claim scientists from Ohio State University. The team behind the feat hope the brain could transform our understanding of neurological disease.

Though not conscious the miniature brain, which resembles that of a five-week-old foetus, could potentially be useful for scientists who want to study the progression of developmental diseases. It could also be used to test drugs for conditions such as Alzheimer’s and Parkinson’s, since the regions they affect are in place during an early stage of brain development.

The brain, which is about the size of a pencil eraser, is engineered from adult human skin cells and is the most complete human brain model yet developed, claimed Rene Anand of Ohio State University, Columbus, who presented the work today at the Military Health System Research Symposium in Fort Lauderdale, Florida.

Scientists create lab-grown spinal cords. Read more

Previous attempts at growing whole brains have at best achieved mini-organs that resemble those of nine-week-old foetuses, although these “cerebral organoids” were not complete and only contained certain aspects of the brain. “We have grown the entire brain from the get-go,” said Anand.

Anand and his colleagues claim to have reproduced 99% of the brain’s diverse cell types and genes. They say their brain also contains a spinal cord, signalling circuitry and even a retina.

The ethical concerns were non-existent, said Anand. “We don’t have any sensory stimuli entering the brain. This brain is not thinking in any way.”

Anand claims to have created the brain by converting adult skin cells into pluripotent cells: stem cells that can be programmed to become any tissue in the body. These were then grown in a specialised environment that persuaded the stem cells to grow into all the different components of the brain and central nervous system.

According to Anand, it takes about 12 weeks to create a brain that resembles the maturity of a five-week-old foetus. To go further would require a network of blood vessels that the team cannot yet produce. “We’d need an artificial heart to help the brain grow further in development,” said Anand.

Several researchers contacted by the Guardian said it was hard to judge the quality of the work without access to more data, which Anand is keeping under wraps due to a pending patent on the technique. Many were uncomfortable that the team had released information to the press without the science having gone through peer review.

Zameel Cader, a consultant neurologist at the John Radcliffe Hospital, Oxford, said that while the work sounds very exciting, it’s not yet possible to judge its impact. “When someone makes such an extraordinary claim as this, you have to be cautious until they are willing to reveal their data.”

3D-printed brain tissue. Read more

If the team’s claims prove true, the technique could revolutionise personalised medicine. “If you have an inherited disease, for example, you could give us a sample of skin cells, we could make a brain and then ask what’s going on,” said Anand.

You could also test the effect of different environmental toxins on the growing brain, he added. “We can look at the expression of every gene in the human genome at every step of the development process and see how they change with different toxins. Maybe then we’ll be able to say ‘holy cow, this one isn’t good for you.’”

For now, the team say they are focusing on using the brain for military research, to understand the effect of post traumatic stress disorder and traumatic brain injuries.

ORIGINAL: The Guardian

Helen Thomson

Tuesday 18 August 2015 

Synthetic Blood Transfusions Are Coming

Cancer-curing Cylon baby blood may still be a fantasy, but with the next two years, two human volunteers will be receiving the very first blood transplants manufactured in a lab, the British National Health Service announced last week.


Technically, what the NHS is calling the world’s first “synthetic blood” is actually biological in origin: It’s produced in vitro by extracting stem cells from the umbilical cords of newborn babies or from adult bone marrow. Placed in the proper chemical environment in the lab, stem cells can be stimulated along a particular developmental pathway that eventually leads to fully-functional red blood cells. Researchers have been developing the technology to manipulate stem cells for years, and now, our tools have advanced to the point that scaling up and producing entire blood bags seems within reach. 

Sure, synthetic blood may sound a bit creepy, but much like lab-grown tissue transplants or replacement organs, it’ll actually be brilliant if we can make it to work. Eventually, hospitals could stockpile huge quantities of the stuff for emergency transfusions, or design batches specifically for patients suffering from sickle-cell anemia and other rare blood disorders. What’s more, having never been inside a human body, lab-grown blood is practically guaranteed to be disease free, and has the potential to dramatically reduce the risk of spreading blood borne diseases like HIV and hepatitis.

For the first human trials, volunteers will be injected with a few teaspoons of the stuff to test for adverse reactions. Test transfusions will also allow scientists to study how long their lab-grown blood cells survive in a human host. So far, preliminary tests show that synthetic red blood cells are biologically comparable, if not identical, to blood cells produced the ol’ fashion way. But biology is full of surprises, and we’ll never know for sure until we try em’ out in humans.

[The Independent]


Follow Maddie on Twitter or contact her at maddie.stone@gizmodo.com



ORIGINAL: Gizmodo

By Maddie Stone
6/28/15

New tech tool speeds up stem cell research

It’s hard to do a good job if you don’t have the right tools. Now researchers have access to a great new tool that could really help them accelerate their work, a tool its developers say “will revolutionize the way cell biologists develop” stem cell models to test in the lab.

Fluidigm’s Callisto system

Fluidigm’s Callisto system

Add caption

Fluidigm’s Callisto system
The device is called Callisto™. It was created by Fluidigm thanks to two grants from CIRM. The goal was to develop a device that would allow researchers more control and precision in the ways that they could turn stem cells into different kinds of cell. This is often a long, labor-intensive process requiring round-the-clock maintenance of the cells to get them to make the desired transformation.

Callisto changes that. The device has 32 chambers, giving researchers more control over the conditions that cells are stored in, even allowing them to create different environmental conditions for different groups of cells. All with much less human intervention.

Lila Collins, Ph.D., the CIRM Science Officer who has worked closely with Fluidigm on this project over the years, says this system has some big advantages over the past:

“Creating the optimal conditions for reprogramming, stem cell culture and stem cells has historically been a tedious and manually laborious task. This system allows a user to more efficiently test a variety of cellular stimuli at various times without having to stay tied to the bench. Once the chip is set up in the instrument, the user can go off and do other things.”

Having a machine that is faster and easier to use is not the only advantage Callisto offers, it also gives researchers the ability to systematically and simultaneously test different combinations of factors, to see which ones are most effective at changing stem cells into different kinds of cell. And once they know which combinations work best they can use Callisto to reproduce them time after time. That consistency means researchers in different parts of the world can create cells under exactly the same conditions, so that results from one study will more readily support and reflect results from another.

In a news release about Callisto, Fluidigm’s President and CEO Gajus Worthington, says this could be tremendously useful in developing new therapies:

“Fluidigm aims to enable important research that would otherwise be impractical. The Callisto system incorporates some of our finest microfluidic technology to date, and will allow researchers to quickly and easily create complex cell culture environments. This in turn can help reveal how stems cells make fate decisions. Callisto makes challenging applications, such as cellular reprogramming and analysis, more accessible to a wide range of scientists. We believe this will move biological discovery forward significantly.”

And as Collins points out, Callisto doesn’t just do this on a bulk level, working with millions of cells at a time, the way the current methods do:

“Using a bulk method it’s possible that one might miss an important event in the mixture. The technology in this system allows the user to stimulate and study individual cells. In this way, one could measure changes in small sub-populations and find ways to increase or decrease them.”

Having the right tools doesn’t always mean you are going to succeed, but it certainly makes it a lot easier.


ORIGINAL: California's Stem Cell Agency. Center for Regeneratie Medicine

kmccormack2014

June 25, 2015

Scientists coax stem cells to form 3-D mini lungs

University of Michigan Health System

Scientists have coaxed stem cells to grow the first three-dimensional mini lungs. Previous research has focused on deriving lung tissue from flat cell systems or growing cells onto scaffolds made from donated organs.

In a study published in the online journal eLife the multi-institution team defined the system for generating the self-organizing human lung organoids, 3D structures that mimic the structure and complexity of human lungs.

Figure 1. Generation of three-dimensional ventral anterior foregut spheroids from endoderm monolayers.

(A) hESCs were differentiated into foregut endoderm by treating cells with 4 days of Activin A (ACTA) followed by 4 days of NOG+SB. (B) Foregut endoderm (NOG+SB) had high expression of the foregut marker SOX2 while the hindgut marker CDX2 was significantly reduced compared to untreated endoderm controls (End). NOG+SB monolayers had high expression of ventral anterior foregut genes NKX2.1 and PAX8 while the posterior foregut marker PDX1 was reduced. The foregut marker HHEX is expressed in the developing liver, biliary system, and thyroid and remained unchanged. (C) The majority of cells in NOG+SB treated cultures were SOX2 positive (green) compared to the control, in which only scattered clusters of cells were SOX2 positive. The scale bar represents 200 µm. (D) hESCs were differentiated into foregut spheroids by treating cells with 4 days of ACTA and then additional 4–6 days of NOG+SB+FGF4+Ch. Representative images of a spheroid in a matrigel droplet are shown as a whole mount image. Scale bar represents 100 µm. (E) Foregut spheroids (NOG+SB+FGF4+Ch) had high expression of the foregut marker SOX2 while the hindgut marker CDX2 was significantly reduced compared to untreated endoderm control (End) (top panel). Spheroids had high expression of anterior foregut genes NKX2.1 and PAX8 while the posterior foregut marker PDX1 was reduced and HHEX was unchanged (bottom panel). *p < 0.05, error bars represent SEM. (F) The majority of cells in foregut spheroids are FOXA2+ (green, left panel) and SOX2+ (white, right panel) and ECAD+ (red, right panel). Scale bar represent 50 µm.

DOI: http://dx.doi.org/10.7554/eLife.05098.003