From organs to muscle tissue: how stem cells are being used in 3D

A Sunday Afternoon on the Island of La Grande Jatte by Georges-Pierre Seurat

When most people think of stem cells, they might conjure up an image of small dots under a microscope. It is hard to imagine these small specs being applied to three-dimensional structures. But like a pointillism painting, such as A Sunday Afternoon on the Island of La Grande Jatte by Georges-Pierre Seurat, stem cells can be used to help build things never thought possible. Two studies demonstrate this concept in very different ways.

MIT engineers have designed coiled “nanoyarn,” shown as an artist’s interpretation here. The twisted fibers are lined with living cells and may be used to repair injured muscles and tendons while maintaining their flexibility. Image by Felice Frankel

A study at MIT used nanofiber coated with muscle stem cells and mesenchymal stem cells in an effort to provide a flexible range of motion for these stem cells. Hundreds of thousands of nanofibers were twisted, resembling yarn and rope, in order to mimic the pattern found in tendons and muscle tissue throughout the body. The researchers at MIT found that the yarn like structure of the nanofibers keep the stem cells alive and growing, even as the team stretched and bent the fibers multiple times.

Normally, when a person injures these types of tissues, particularly around a major joint such as the shoulder or knee, it require surgery and weeks of limited mobility to heal properly. The MIT team hopes that their technology could be applied toward treating the site of injury while maintaining range of motion as the newly applied stem cells continue to grow to replace the injured tissue.

In an article, Dr. Ming Guo, assistant professor of mechanical engineering at MIT and one of the authors of the study, was quoted as saying,

“When you repair muscle or tendon, you really have to fix their movement for a period of time, by wearing a boot, for example. With this nanofiber yarn, the hope is, you won’t have to wearing anything like that.”

Their complete findings were published in the Proceedings of the National Academy of Sciences (PNAS).

Researchers in Germany have created transparent human organs using a new technology that could pave the way to print three-dimensional body parts such as kidneys for transplants. Above, Dr. Ali Ertuerk inspects a transparent human brain.
Photo courtesy of Reuters.

In a separate study, researchers in Germany have successfully created transparent human organs, paving the way to print three-dimensional body parts. Dr. Ali Erturk at Ludwig Maximilians University in Munich, with a team of scientists, developed a technique to create a detailed blueprint of organs, including blood vessels and every single cell in its specific location. These directions were then used to print a scaffold of the organ. With the help of a 3D printer, stem cells, acting like ink in a printer, were injected into the correct positions to make the organ functional.

Previously, 3D-printed organs lacked detailed cellular structures because they were based on crude images from computer tomography or MRI machines. This technology has now changed that.

In an article, Dr. Erturk is quoted as saying,

“We can see where every single cell is located in transparent human organs. And then we can actually replicate exactly the same, using 3D bioprinting technology to make a real functional organ. Therefore, I believe we are much closer to a real human organ for the first time now.”

Using 3D printer to develop treatment for spinal cord injury

3d-printed-device

3D printed device

Spinal cord injuries (SCIs) affect approximately 300,000 Americans, with about 18,000 new cases occurring per year. One of these patients, Jake Javier, who we have written about many times over the past several years, received ten million stem cells as part of a CIRM-funded clinical trial and a video about his first year at Cal Poly depicts how these injuries can impact someone’s life.

Currently, there is nothing that completely reverses SCI damage and most treatment is aimed at rehabilitation and empowering patients to lead as normal a life as possible under the circumstances. Improved treatment options are necessary both to improve patients’ overall quality of life, and to reduce associated healthcare costs.

Scientists at UC San Diego’s School of Medicine and Institute of Engineering in Medicine have made critical progress in providing SCI patients with hope towards a more comprehensive and longer lasting treatment option.

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Prof. Shaochen Chen and his 3D printer

In a study partially funded by CIRM and published in Nature Medicine, Dr. Mark Tuszynski’s and Dr. Shaochen Chen’s groups used a novel 3D printing method to grow a spinal cord in the lab.

Previous studies have seen some success in lab grown neurons or nerve cells, improving SCI in animal models. This new study, however, is innovative both for the speed at which the neurons are printed, and the extent of the neuronal network that is produced.

To achieve this goal, the scientists used a biological scaffold that directs the growth of the neurons so they grow to the correct length and generate a complete neuronal network. Excitingly, their 3D printing technology was so efficient that they were able to grow implants for an animal model in 1.6 seconds, and a human-sized implant in just ten minutes, showing that their technology is scalable for injuries of different sizes.

When they tested the spinal cord implants in rats, they found that not only did the implant repair the damaged spinal cord tissue, but it also provided sustained improvement in motor function up to six months after implantation.

Just as importantly, they also observed that blood vessels had infiltrated the implanted tissue. The absence of vascularized tissue is one of the main reasons engineered implants do not last long in the host, because blood vessels are necessary to provide nutrients and support tissue growth. In this case, the animal’s body solved the problem on its own.

In a press release, one of the co-first authors of the paper, Dr. Kobi Koffler, states the importance and novelty of this work:

“This marks another key step toward conducting clinical trials to repair spinal cord injuries in people. The scaffolding provides a stable, physical structure that supports consistent engraftment and survival of neural stem cells. It seems to shield grafted stem cells from the often toxic, inflammatory environment of a spinal cord injury and helps guide axons through the lesion site completely.”

In order to make this technology viable for human clinical trials, the scientists are testing their technology in larger animal models before moving into humans, as well as investigating how to improve the longevity of the neuronal network by introducing proteins into the scaffolds.

 

 

3D printing blood vessels: a key step to solving the organ donor crisis

About 120,000 people in the U.S. are on a waiting list for an organ donation and every day 22 of those people will die because there aren’t enough available organs. To overcome this organ donor crisis, bioengineers are working hard to develop 3D printing technologies that can construct tissues and organs from scratch by using cells as “bio-ink”.

Though each organ type presents its own unique set of 3D bioprinting challenges, one key hurdle they all share is ensuring that the transplanted organ is properly linked to a patient’s  circulatory system, also called the vasculature. Like the intricate system of pipes required to distribute a city’s water supply to individual homes, the blood vessels of our circulatory system must branch out and reach our organs to provide oxygen and nutrients via the blood. An organ won’t last long after transplantation if it doesn’t establish this connection with the vasculature.

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Digital model of blood vessel network. Photo: Erik Jepsen/UC San Diego Publications

In a recent UC San Diego (UCSD) study, funded in part by CIRM, a team of engineers report on an important first step toward overcoming this challenge: they devised a new 3D bioprinting method to recreate the complex architecture of blood vessels found near organs. This type of 3D bioprinting approach has been attempted by other labs but these earlier methods only produced simple blood vessel shapes that were costly and took hours to fabricate.  The UCSD team’s home grown 3D bioprinting process, in comparison, uses inexpensive components and only takes seconds to complete. Wei Zhu, the lead author on the Biomaterials publication, expanded on this comparison in a press release:

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Wei Zhu

“We can directly print detailed microvasculature structures in extremely high resolution. Other 3D printing technologies produce the equivalent of ‘pixelated’ structures in comparison and usually require … additional steps to create the vessels.”

 

As a proof of principle, the bioprinted vessel structures – made with two human cell types found in blood vessels – were transplanted under the skin of mice. After two weeks, analysis of the skin showed that the human grafts were thriving and had integrated with the mice’s blood vessels. In fact, the presence of red blood cells throughout these fused vessels provided strong evidence that blood was able to circulate through them. Despite these promising results a lot of work remains.

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Microscopic 3D printed blood vessel structure. Photo: Erik Jepsen/UC San Diego Publications

As this technique comes closer to a reality, the team envisions using induced pluripotent stem cells to grow patient-specific organs and vasculature which would be less likely to be rejected by the immune system.

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Shaochen Chen

“Almost all tissues and organs need blood vessels to survive and work properly. This is a big bottleneck in making organ transplants, which are in high demand but in short supply,” says team lead Shaochen Chen. “3D bioprinting organs can help bridge this gap, and our lab has taken a big step toward that goal.”

 

We eagerly await the day when those transplant waitlists become a thing of the past.

Five Cool Stem Cell Technologies to Tell Your Friends

As a former stem cell scientist turned science communicator, I love answering science questions no matter how complicated or bizarre. The other day my friend asked me about what CRISPR was and how scientists were using it on stem cells to help people. This got me thinking that it would be cool to do a blog on some of the latest stem cell technologies that are changing the way we do science and ultimately how we treat patients.

So in the spirit of sharing knowledge and also giving you some interesting conversation points at your next dinner party, here are five stem cell technologies that I think are pretty awesome. (As a disclaimer: this isn’t a top 5 list. I picked a few recently published studies that I thought were worth mentioning.)

1) Need a body part? Let me print that for you.

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3D printed ear. (Wake Forest University)

Scientists from Wake Forest University have developed technology to make custom-made living body parts by 3D-printing stem cells onto biodegradable scaffolds. The stem cells are printed in a hydrogel solution using a special 3D printer they call ITOP. This printer makes it possible for the printed stem cells to develop into life-sized tissues and organs that have built-in microchannels that allow blood, oxygen and other nutrients to flow through. Using the ITOP technology, the team was able to generate segments of jawbone, an ear, and muscle tissue. We wrote a blog about this fascinating technology, so check it out if you’re thirsty for more details.

 2) Bio-bots controlled by light

When you think robots, you think machines and metal. But what if the robot was made out of human cells? Crazy? Not even. Scientists from the University of Illinois have made what they called “bio-bots” or tiny machines “powered by biological components.” They printed muscle cells onto flexible skeletons in the shape of rings (see GIF). The muscle cells are engineered to have light sensitive switches, so when they are exposed to light, they contract like normal muscles do. The beauty of bio-bots is that they “can sense, process, and respond to dynamic environmental signals in real time, enabling a variety of applications.” Some of these applications could include bio-bots made up of other types of tissue (brain, heart, etc.) and general use for disease research. Story credit goes to Megan Thielking’s Morning Rounds for STATnews.

Bio-bots composed of muscle cells are powered by light. (University of Illinois)

Bio-bots composed of muscle cells are powered by light. (University of Illinois)

3) New way to track stem cells using MRI

Scientists from the UC San Diego School of Medicine have developed a new way to track cells in the body using magnetic resonance imaging (MRI). In a CIRM-funded study, the scientists made a new Fluorine-based chemical tracer that is taken in by the cells of interest. When these cells are imaged with MRI, the tracer gives off a bright and easily detectable signal. According to MNT news who covered the story, “the work is expected to enhance the progress of treatments involving stem cells and immune cells, as it will give researchers a clear picture of how cells behave after being introduced to the body.”

 4) Engineering cells to fight cancer

Genomic modification of human stem cells by gene editing methods such as CRISPR is not a novel concept, but the technology continues to evolve at record pace and is worth mentioning. You can think of CRISPR as molecular scissors that can remove disease-causing mutations in a person’s DNA. Scientists can repair genetic mutations in human stem cells and other cell types and then use these repaired cells to replace diseased or damaged tissue or to perform therapeutic functions in patients. An article by Antonio Regalado at MIT Technology Review nicely summarizes how genetically engineered immune cells are saving the lives of cancer patients. These immune cells are engineered to recognize cancer cells (which are normally expert at evading the immune system) and when they are transplanted into cancer patients, they attack and kill off the cancer pretty effectively.

5) One day, stem cells will help the blind see

Artistic representation of the human eye. (Dr. Kang Zhang, Dr. Yizhi Liu)

Artistic representation of the human eye. (Dr. Kang Zhang, Dr. Yizhi Liu)

Blindness is a big problem and stem cells are considered a promising therapeutic strategy for restoring sight in patients suffering from diseases of blindness. We covered two recent discoveries in last week’s round-up, but it never hurts to mention them again. One study from UC San Diego Health treated children suffering from cataracts. They removed the cataracts and stimulated the native stem cells in their eyes to produce new lens tissue that was able to improve their vision. The other study generated different eye parts in a dish using reprogrammed human induced pluripotent stem cells or iPS cells. They generated corneas from iPS cells and transplanted them into blind rabbits and were successful in restoring their vision. Hopefully soon stem cell technologies will advance through the clinic and provide new treatments to cure patients who’ve lost their sight.

Meet ITOP: A One Stop Shop for 3D Printing Body Parts

“They have managed to create what appears to be the goose that really does lay golden eggs!”

That was how UK surgeon Martin Birchall described it to BBC News. The goose in this case is a 3D bioprinter, and the golden eggs are the human sized tissues that the bioprinter successfully constructed. This breakthrough for the field of tissue engineering was reported on Monday in Nature Biotechnology by a research team led by Anthony Atala, director of Wake Forest Institute for Regenerative Medicine.

Bioprinting: yes, it’s actually a thing

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The 3D bioprinting process. Image: Wake Forest, Nature Biotechnology

To some, printing human body parts may sound like a far-fetched story torn from a science fiction novel but, in reality, development of bioprinters has been underway for a number of years. The bioprinting process isn’t all that different from an inkjet printer except a mixture of gelatin, or hydrogel, and living cells is used as the “ink” to incrementally build a defined  biological structure.

As amazing as this technology is, it has met some limitations. Printing cells into 2D shapes and small 3D structures is doable but a lack of structural stability limits building more complex, human-scale tissues. Also, because oxygen and nutrients can only diffuse about 0.004 inches through living tissue, the cells located inside a large bioprinted structure have trouble with long-term survival (check out yesterday’s blog for a mind-blowning story about one lab’s use of cotton candy to deal with this diffusion issue). These challenges have to be overcome before 3D bioprinting can be used for the repair and replacement of human-sized tissues and organs.

Enter ITOP
That’s where the Atala team’s Integrated Tissue-Organ Printer (ITOP), developed over ten years, comes into the picture. For better structural stability, the printer is configured to deliver the cell/hydrogel “bio-ink” within a stronger type of gel, with the fancy name Pluronic F127, that helps the printed cells maintain their shape during the printing process. Afterward, the Pluronic F127 scaffolding mold is simply washed away from the bioprinted tissue.

ITOPcloseup

Close-up view of ITOP: a 3D bioprinter. Image: Wake Forest, Nature Biotechnology.

To ensure adequate oxygen diffusion into the bioprinted tissue, a biodegradable polymer, PCL, is dispensed from the printer at regular intervals; this creates microchannels as stand-ins for blood vessels to help oxygen and nutrients readily reach the interior areas of the tissues.  As a bonus, PCL takes about 2 years to biodegrade, providing long term stability.

To prove these innovations of the ITOP actually work, the team built three different tissues: a jawbone fragment, an ear, and muscle.

The making of a jawbone
For reconstruction of the jawbone, a 3D computer model was generated from actual CT scan data of a human jaw with a missing piece of bone – something that might be seen in a traumatic injury to a combat soldier (the work is funded in part by the Armed Forces Institute for Regenerative Medicine). That data was fed into the ITOP with coordinates of the precise printing pattern necessary to rebuild the shape of the jaw fragment. In this case, printing was carried out using human amniotic fluid stem cells (AFSC). With the right cues, these stem cells readily specialize into osteogenic, or bone-forming, cells.

Sure enough, 28 days after being cultured in liquid nutrients containing bone-promoting factors, the surface of the bioprinted human jaw showed calcium deposits, the tell-tale signs of bone formation. How would bioprinted bone fare in a living mammal? To find out, the researchers transplanted small discs of AFSC fabricated bone into a bone defect in mice. After 5 months, the transplanted bone was thriving with plenty of blood vessels and no necrosis, or cell death, inside the bone.

The implications of this bone study are pretty cool. In an interview with BBC news, Atala envisions a not so distant future clinical scenario:

“We’d bring the patient in, do the imaging and then we would take the imaging data and transfer it through our software to drive the printer to create a piece of jawbone that would fit precisely in the patient.”

Vincent van Gogh could have used this technology
But the possibilities don’t end with bone. Next, the team tested the ITOP’s talents at building the complex shapes of the outer human ear. In this case, the printer was loaded up with cartilage-producing cells called chondrocytes. The authors posted a fascinating video of the ear bioprinting process in their online publication.

ear_wakeforest

A human-sized bioprinted ear. Image: Wake Forest, Nature Biotechnology

Image: Wake Forest, Nature Biotechnology.

After five weeks in liquid nutrients, a matrix of cartilage had grown throughout the ear. And to look at tissue growth in an animal, the ear was implanted under the skin of the mice. A couple of months after implantation, even more cartilage had formed and the shape of the ear was intact.

But wait there’s more: printing skeletal muscle
Since both the jaw bone and ear cartilage represent hard tissues, the team sought to reconstruct muscle, a soft tissue, with the ITOP. Muscle-forming cells, or myoblasts, were printed to mimic the muscle fiber bundles seen in native skeletal muscle. After growing a week in the lab under conditions that stimulate muscle cell formation, the muscle-like fibers were implanted into rats. Two week after implantation, the bioprinted muscle had not only grown into well-organized muscle fibers, they also were functional in that they were responsive to electrical stimulation.

3D bioprinting: getting closer to reality
Atala is the first to admit that a lot more testing is needed to safely bring this technology into a clinical setting for human use. But as he states in a Wake Forest press release, the ITOP brings 3D bioprinting a step closer to reality:

“This novel tissue and organ printer is an important advance in our quest to make replacement tissue for patients. It can fabricate stable, human-scale tissue of any shape. With further development, this technology could potentially be used to print living tissue and organ structures for surgical implantation.”

 

 

Students use a 3D printer to sink their teeth into stem cell research

Student winners

L-R Alan Tan, Sid Bommakanti, Daniel Chae – prize winning science students

A 3D printer, some old teeth, and some terrific science were enough to help three high school students develop a new way of growing bone and win a $30,000 prize in a national competition.

The three teamed up for the Siemens Competition in Math, Science & Technology, which bills itself as “the nation’s premier research competition for high school students”.

The trio includes two from the San Francisco Bay area, where we are based; Sid Bommakanti from Amador Valley High School in Pleasanton, and Alan Tan, from Irvington High School in Fremont. The third member of the team, Daniel Chae, goes to Thomas Jefferson High School for Science and Technology in Alexandria, Virginia.

The three used mesenchymal stem cells – which are capable of being turned into muscle, cartilage or bone – which they got from the dental pulp found in wisdom teeth that had been extracted.

In a story posted on the KQED website Tan says they thought it would be cool to take something that is normally thrown away, and recycle it:

“When we learned we could take stem cells from teeth—it’s actually part of medical waste—we realized could turn this into bone cells,”

The students used a 3D printer to create a kind of scaffold out of a substance called polylatctic acid – it’s an ingredient found in corn starch or sugar cane. The scaffold had a rough surface, something they hoped would help stimulate the dental pulp to grow on it and become bone.

That’s what happened. The students were able to show that their work produced small clusters of cells that were growing on the scaffold, cells that were capable of maturing into bone. This could be used to create dental implants to replace damaged teeth, and, according to Alan Tan, to repair other injuries:

“We used dental pulp stem cells so that we could regenerate bones in various parts of our body so for example we could fix bones in your jaw and tibia and other places.”

The beauty of this approach is that the scaffold and bone could be implanted in, say, the mouth and then as the scaffold disintegrates the new bone would be left in place.

While they didn’t take the top prize (a $100,000 scholarship) they did have to see off some serious competition from nearly 1,800 other student project submissions to win a Team scholarship award.

The students say they learned a lot working together, and encouraged other high school students who are interested in science to take part in competitions like this one.

Sid Bommakanti “Both me, Alan and our other partner are interested in medicine as a whole and we wanted to make an impact on other people’s lives.”

Alan Tan: “I would say get into science early. Don’t be afraid to put yourself out there and talk to professors, talk to people, competitions like this are beneficial because they encourage students to get out there and interact with the real world.”

CIRM is helping students like these through its Stem Cell Education Portal,  which includes the materials and resources that teachers need to teach high school students about stem cells. All the materials meet both state and federal guidelines.

 

 

3D Printing Cells with DNA Velcro

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The complex, 3D micro-anatomy of the human liver. (Image source: WikiMedia Commons)

One of the Holy Grails of stem cell research is growing body parts to replace those damaged by disease or injury. Enormous strides have been made in a key first step: mastering recipes for maturing stem cells into various specialized cell types. But a lawn of, say, liver cells in a petri dish is not a functioning liver. Organs have complex, three-dimensional structures with intricate communication between multiple cell types.

Scientists are actively devising methods to overcome this challenge. For instance, cultivating cells onto biological scaffolds help mold the cells into the shape of a particular organ or tissue. And retooled 3D printers using “bio ink” can seed layers of different cells onto these scaffolds to create specified structures.

This week, a UCSF team added an ingenious new tool to this tissue engineering tool kit.  As reported on Monday in Nature Methods, the lab of Zev Gartner took advantage of DNA’s Velcro-like chemistry to build layers of different cell types in a specified pattern.

DNA – it’s not just for genetics anymore

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A DNA fragment is made of two complimentary strands that bind together with high specificity. (Image source: Visionlearning)

DNA is a molecule made of two thin strands. Each strand is specifically attracted to the other based on a unique sequence of genetic information. So if two strands of a short DNA fragment are peeled apart, they will only rejoin to each other and not some other fragment with a different sequence.  While DNA usually resides in the nucleus of a cell, the team worked out a method to temporarily attach copies of a strand of DNA on the outside of, let’s call it, “cell A”. The opposite strand of that DNA fragment was attached to “cell B”. When mixed together the two cells became attached to each other via the matching DNA sequences. Other cells with different DNA fragments floated on by.

The screen shot below from a really neat time-lapse video, which accompanies the research publication, shows how a rudimentary 3D cell structure could be built with a series of different cell-DNA fragment combinations. In this case, the team first attached DNA fragments onto a petri dish in a specific pattern. At the thirty-second mark in the video, you can see that cells with matching DNA fragments have attached to the DNA on the dish.

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This video demonstrates the assembly of 3D cell structures with the help of DNA “Velcro” (image source: Todhunter et al. Nature Methods 2015 Aug 31st)

The new technique, dubbed DNA programmed assembly of cells (DPAC), opens up a lot possibilities according to Gartner in a UCSF press release:

 “We can take any cell type we want and program just where it goes. We can precisely control who’s talking to whom and who’s touching whom at the earliest stages. The cells then follow these initially programmed spatial cues to interact, move around, and develop into tissues over time.”

The Quest still continues with possible victories along the way

 Of course, this advance is still a far cry from the quest for whole organs derived from stem cells. The cell assemblies using DPAC can only be grown up to about 100 microns, the thickness of a human hair. Beyond that size, the innermost cells get starved of oxygen and nutrients. Gartner says that obstacle is a current focus in the lab:

“We’re working on building functional blood vessels into these tissues. We can get the right cells in the right positions but haven’t figured out how to perfuse them with blood or a substitute efficiently yet.”

In the meantime, building these small 3D “organoids” from stem cells certainly could be put to good use as a means to test drug toxicity on human tissue or as a way to study human disease.

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