Body builders: using stem cells to cultivate organs.

The field of tissue engineering has long been fraught with hope and hype. For the past several decades, laboratory scientists have pursued the ambitious goal of growing new organs and tissues--a heart, say, or a piece of spinal cord--that a surgeon could transplant into a patient. This capability could potentially solve the chronic shortage of donor organs while offering physicians new ways of treating patients with diseased and damaged tissues, such as knees or hips ravaged by arthritis. Easier said than done.

The general concept behind tissue engineering is relatively simple. Take a biodegradable polymer scaffold, mold it into a particular anatomical shape, and seed it with cells of the sort of tissue needed. As the cells proliferate and form into new tissue, the scaffold erodes, leaving behind the desired body part.

Until recently, researchers have relied on cells extracted from patients and cultured in the lab. However, these cells have many limitations. Now, more and more scientists are turning to stem cells because they can proliferate rapidly and grow into a variety of cell types.

For relatively simple organs, including the bladder, and tissues, such as skin, the older strategy appears to work fine. But building complex structures, such as a heart, would require removing a sample of cells from the patient's organ, isolating each type of heart cell, growing the cells separately in the lab, and seeding them on a scaffold in a precise configuration. Moreover, organize themselves into three-dimensional structures is difficult.

"If a patient comes in with heart failure, you're not going to go in and take out a piece of his heart tissue," says Anthony Atala, director of the Regenerative Medicine and Tissue Engineering Institute at Wake Forest University in Winston-Salem, N.C. Surgically removing heart tissue from a patient to grow a new piece of heart in the lab, he says, would put the already-weak patient at risk.

You might extract a stem cells, however, either from the patient's bone marrow or from an embryonic or fetal source. Not only do these primitive cells provide tissue engineers with a new source of cells to repair or grow organs, they might also be coaxed into generating more-complex tissues and organs than have been possible with differentiated cells.

What's more, mixing stem cells with polymer materials could be a potent strategy for repairing damaged tissues. Scientists have tried with some success to use injections of stern cells to repair injured spinal cords in lab animals. A supporting structure might improve the outcome. "Even the best stem cells are going to need a template to allow the cells to take hold;' says Evan Snyder, a developmental biologist at the Burnham Institute in La Jolla, Calif.

By designing new scaffolds that can interact with stem cells, researchers are working to mimic the way tissues and organs naturally develop in the body. Says chemical engineer Jennifer Elisseeff of Johns Hopkins University in Baltimore, "Stem cells have really given the field of tissue engineering an extra push."

FEELING THE WAY Fantastic orchestration underlies tissue development; trying to recapitulate it in the lab is a daunting undertaking. That challenge requires designing new materials that are strong enough to support and guide cells, that provide specific biological signals that make the cells behave in certain ways, and that degrade at the appropriate rates.

Over the years, researchers have made great strides in designing increasingly sophisticated scaffolds, such as polymers reinforced with aluminum for supporting bone and polymers that degrade in synchrony with new tissue growth (SN: 4/26/03, p. 261). Now, tissue engineers are turning their attention to more sophisticated cells.

Last fall, bioengineer Robert anger and his colleagues at the Massachusetts Institute of Technology, (MIT) showed that growing human embryonic stem cells on three-dimensional scaffolds could induce the cells to differentiate and form multiple tissue types. The researchers created several polymer scaffolds and added to each one a growth factor associated of tissue. Two weeks seeding of scaffolds with stem cells, either cartilage, livers, or nervous tissue formed on each of the scaffolds. In the liver sample, a network of blood vessels also emerged.

"Seeing the structures grow was very exciting" says Shulamit Levenberg, a member of the MIT group.

The researchers then implanted the tissue-containing scaffolds under skin on the backs of mice. Not only did the stem cells continue to differentiate and form into their fated tissue, blood vessels from the mice started making connections with the tissue growing inside the scaffolds. The researchers described the experiment in the Oct. 13, 2003 Proceedings of the National Academy of Sciences.

Although the growth factors played a role in pushing the cells toward one tissue type or another, the scaffold was critical to the differentiation. In the same experiment, the researchers also grew stem cells on a soft gel material. When compared with cells on the harder scaffolds, the cells on the gel showed only early signs of differentiation.

Langer's team isn't the only one to have demonstrated the power of the scaffold. Snyder, for instance, seeded polymer scaffolds with neural stem cells and then implanted the complexes in the stroke-damaged brains of mice.

Without any chemical cues, the cells spontaneously differentiated into neurons and other brain cells called oligodendrocytes. The cells extended throughout the polymer fibers making up the scaffold. Meanwhile, neuronal fibers and new blood vessels from the mice infiltrated the regenerating brain tissue.

Christopher Chen at Johns Hopkins University conducted a series of experiments to show how mechanical cues alone can trigger stem cells to differentiate. By modifying a plastic chip's surface the researchers created arrays of different-size islands such that a cell placed on an island could not expand beyond its edge. The cells that had little space remained spherical, while those with more area available flattened out.

In December, at the American Society for Cell Biology's annual meeting in San Francisco, the Johns Hopkins team reported that flattened stem cells turned into bone, and cells that remained round turned into fat. "The cells seemed to detect their own structure;' says Chen.

A gene called Rho appears to be at the heart of this phenomenon. Chen and his colleagues determined that when stem cells are stretched out, the gene turns on and causes the cells to build internal structures that increase tension. This tension then steers the cells toward the bone pathway.

MIX AND MATCH By emulating the body's coordinated use of multiple biological cues, researchers are learning how to get stem cells to do even more of their bidding. Tissue engineers can selectively place different growth factors in different parts of a polymer scaffold, for example, to push a population of stem cells to form multiple layers of different tissue types.

Elisseeff and her coworkers at Johns Hopkins have developed an approach for creating such multilayers. Instead of placing cells on a solid scaffold, the researchers mix the cells into a liquid polymer. When exposed to ultraviolet light, a photosensitive chemical in the liquid causes the material to harden and encapsulate the cells.

To create multiple layers, the researchers mix stem cells and a growth factor into a layer of the liquid polymer and then partially solidify the material with a dose of UV light. A second polymer layer is added with the same stem ceils but a different chemical cue. A final beam of light cinches together the two layers. The researchers are striving for implants that have adjacent layers of cartilage and bone.

Because cartilage is slippery, having a layer of bone underneath it could help surgeons anchor the cartilage to a patient's joint. Currently, surgeons have difficulty- integrating replacement cartilage taken from a donor, with the surrounding tissue, says Elisseeff.

Besides the liquid-polymer approach, tissue engineers are pursuing a host of strategies for guiding stem cell differentiation at different points in a scaffold. One tactic encloses growth factors in degradable microspheres and places them in different locations of the polymer material.

Simply attaching growth factors to the polymer fibers themselves is another approach. "Depending on where the stem cells fall on the scaffold, that's what they become" says Atala. His lab has used this method to direct uniform stem cells to create layers of muscle on top of cartilage. Atala hopes eventually to use this approach for engineering replacement digits for patients who have lost a finger or toe, for example.

To gain yet more control over the patterning of the cues, Atala and others are experimenting with three-dimensional printers that can build scaffolds by depositing layer upon layer of the polymer material, while incorporating specified types and amounts of growth factors at explicit locations.

And most recently, in a Jan. 22 online posting at Scienceexpress, scientists at Northwestern University in Evanston, Ill., devised a way to control the type and density of chemical signals adorning each polymer fiber. The researchers designed protein building blocks customized to self-assemble into nanoscale fibers with the desired chemical cues on their surfaces. Three-dimensional gel-like scaffolds made from these fibers then guided growth of neural stem cells and triggered them to differentiate into neurons.

TRUE HEALING Developmental biologists are only beginning to identify the menagerie of chemical signals that trigger stem cells to differentiate and form new tissue. By harnessing the power of these cells and their signals, researchers might induce the body to heal on its own. David Mooney, a bioengineer at the University of Michigan in Ann Arbor, envisions a day when tissue engineers will rig their scaffolds with a battery of growth factors that activate in a specific sequence to get the desired effect.

"Cells don't differentiate based on a single piece of information:' Mooney notes. "The signals are very dynamic, so if we can control their timing and sequence, we can mimic real tissue development."

Or, says Snyder, stem cells placed in a more cleverly designed scaffold without any chemical cues could awaken the body's own capacity for regeneration. For example, he and his colleagues fabricated scaffolds with intricate, multilayered and seeded them with neural stem cells. When they implanted these structures into excised regions of rats' spinal cords, the stem cells provided the appropriate cues to trigger the animals' nervous systems to repair themselves.

In this particular experiment, one of Snyder's colleagues, Erin Lavik, now at Yale University, designed a polymer scaffold to emulate the microstructures in spinal cord. "The actual chemistry involved in putting it together took 2 years," says Snyder. "It was a real tour de force."

The scaffold consisted of two layers--an internal layer with densely packed pores for hosting the stem cells and an outer layer with long pores oriented in different directions for transporting fluids and guiding nerve cell growth. The researchers removed a section of the rats' spinal cords and replaced each section with the stem cell-bearing scaffold.

When placed in the rats' severed spinal cords, the stem cells remained in their primitive state inside the scaffold and recruited nerve cells from adjacent tissues. What's more, the implant prevented scar tissue formation, reduced inflammation, and rescued nearby cells on the brink of dying.

Snyder hopes that the tools of tissue engineering will enable physicians to be more proactive in treating stroke and spinal cord injury. Today, when a person with a spinal cord injury is admitted to the hospital, physicians can assess the damage, but they have few ways of intervening. They can give the patient steroids to reduce inflammation. "But essentially, you wait and see what functions come back," says Snyder.

In the future, a physician might capitalize on the window of opportunity in the days following an injury "when the terrain is still quite fertile for self-repair," says Snyder. That's when a surgeon might implant a scaffold seeded with stem cells that could fill in the once-irreparable defect, he suggests.

Many in the field of tissue engineering share this powerful vision. As they see it, the marriage of materials and stem cells could open the way to a renewable supply of healthy tissues and organs.