Developmental biologists, at least those interested in mammalian development, will tell you that the early 1970s were an exceptionally stimulating time, and all because of the gift that Roy Stevens had given their field. The mice of Strain 129 and their bizarre yet fascinating teratomas were a hot item, and a small but fervent coterie of aspiring postdocs and rising and already-risen embryologists wanted this rare mouse in their labs. They delighted in the chance to study the stem cells inside a mouse’s gonadal tumor and watch them grow and differentiate into all types of tissue. This preoccupation was possible because scientists were learning a new trick, namely how to keep stem cells from mouse teratomas alive in a petri dish.

“I was doing it for the very reason that so many other people were doing it,” describes John Gearhart, who in the early ’70s was a postdoc at what now is the Fox Chase Cancer Center in Philadelphia. “Finally we had an experimental system in mammalian embryology where we had a pluripotent cell sitting in a dish, and we could say, look, we’re getting nerve cells and muscle cells and others. Thanks to what Barry Pierce did, we knew that a single cell was responsible for forming all of these other cell types.”

Working at MIT was a cell biologist M.D. who found the prospect of exploring mouse teratomas so alluring that at age forty-six he had actually switched fields for this very purpose. He was Howard Green, another gifted Canadian-born scientist. Shortly before joining MIT’s biology department in 1970, while still a professor at New York University School of Medicine, he had invented a novel method for assigning a human gene to its respective chromosome, a practice seized upon by medical geneticists. Yet despite this achievement, “I thought it would be more interesting to study cell differentiation in a teratoma,” Green recalls, even though he “had no fixed idea of how to proceed.” A cordial, trim, neatly attired gentleman who prefers not to retire quite yet, Green presently holds the position of George Higginson Professor of Cell Biology at Harvard Medical School.

Because Howard Green was the sort of scientist who liked to directly connect with other scientists whose work intrigued him, one cold day in the late fall of 1972 he and James Rheinwald, one of his graduate students, made the long drive north to the Jackson Laboratory on Mount Desert to pay Roy Stevens a visit. They soon returned to MIT with a few of Stevens’s popular tumor-bearing mice. While Green had no precise plan for his research, he was curious to see for himself the assortment of cell types that a teratoma might yield. It wasn’t long after Rheinwald had sacrificed a mouse, minced its tumor, and set the cells growing in a culture dish that one certain cell claimed Green’s attention, because its identity stumped him. He was familiar with many types of cells, but he’d never seen the likes of this one before. It was flat and compact and nothing like a fibroblast (the predominant cell in connective tissue) with its irregular shape, or a red blood cell with its doughnut-like concave center.

Rheinwald tried separating cells of this type away from other teratoma cells, but they wouldn’t grow alone. They seemed to need the support of other cells to flourish. Biologists had become wise to the fact that for cells to multiply in the artificial setting of a culture dish half as well as they did in the body, they often required the

proximity of feeder cells, cells that lend support to other cells. One had only to appreciate how densely packed the body is with cells to realize that it wasn’t natural for one type of cell to grow on its own, in obscurity, without neighbors. So Rheinwald thawed and made use of 3T3 feeder cells, a cell line that Green had developed in the early ’60s while at NYU. (When a cell and its progeny are made to divide and therefore “grow” indefinitely, the result is a cell line, an invaluable resource that provides researchers with a near limitless and accessible source of cells.) These 3T3 cells were fibroblasts from the connective tissue of fetal mice, and labs clear around the globe had come to depend on them because of how they boosted the growth of other cells. Green’s nickname for them—3T3s—referred to a necessary requirement for their growth. They had to be transferred to new flasks three times a week at a density of about 300,000 cells per dish.

When added to other cells, 3T3s first were exposed to X rays to prevent their own growth in culture—that is, in a dish filled with specially made media. This done, Rheinwald commingled the feeder cells with the teratoma’s mystery cell—and the strategy worked. The nameless cell began to flourish, indicating that the added feeder cells released a protein that the mystery cell found nourishing.

Now that their cell was growing so nicely, dividing and proliferating happily, Green and Rheinwald had a much better chance of learning its identity. They already had discussed its resemblance to an epithelial cell. Found everywhere in the body and assuming many different shapes and structures, epithelial cells share the common motif that they form surfaces, whether the body’s outer surfaces or the interior surfaces of numerous cavities and passageways. Flat, cuboid, or cylindrical, they pave the surfaces of tongue, cheek, esophagus, intestine, urinary tract, the lung’s alveoli, and the ear and nose canals, to name only a few of the places they reside. To compensate for the constant wear and tear they take, Nature has made these cells highly regenerative.

Their cell’s epithelial nature really spoke to Rheinwald. After

getting his bachelor’s degree from the University of Illinois, and before coming to Harvard Medical School, he had spent a year at the University of Wisconsin tending fast-dividing fibroblast cells, which had left him puzzling over why epithelial cells failed to similarly thrive when put in the same basic medium. With Green’s approval, Rheinwald decided to devote his doctoral thesis to their unidentified epithelial cell, and maybe this time around he would determine why its growth requirements were different from a fibroblast’s. Cells, scientists were realizing, were as finicky as people; culture conditions that suited one type of cell didn’t necessarily suit another.

The question that Green and Rheinwald were left with was, which of the many classes of epithelium did their mystery cell belong to? One spring day in 1971, some unexpected assistance arrived in the form of Burton Goldberg, a colleague of Green’s from NYU. Glancing through a microscope at colonies of their cells, Goldberg exclaimed, “Why, that’s a beautiful stratified squamous epithelium!” The kind of epithelium he was referring to forms the outer barrier layer of the skin (the epidermis), the cornea, the mouth, and the esophagus. An exceedingly durable tissue, it is composed of keratinocytes, cells that synthesize a tough protein called keratin, and it was the lavish layering of stratified keratinocytes that Goldberg’s eagle eyes had spotted.

Progress! But now Green and Rheinwald were left to wonder which bodily surface their keratinocyte cells actually belonged to. Skin? Cornea? Mouth? Esophagus? It remained to be seen whether keratinocytes from these areas, when placed in the company of 3T3 feeders, would grow as well as their teratoma-derived cells. They decided to first test keratinocytes from skin, which were easily obtained. Rheinwald simply contacted his wife’s obstetrician at the Boston Lying-In Hospital, who set aside the circumcised foreskin of a newborn, and during a free moment Rheinwald picked up the sample, which lay ensconced in a plastic tube inside a tin can, and transported it back to MIT on the subway—not without a few thoughts about how odd a biologist’s life sometimes could be. In the lab, he

set about disassociating the skin cells and placed them in a dish together with Green’s hearty 3T3 cells, eager to see whether they would survive and multiply. The year was 1974, and the day, Rheinwald recalls, was September 10—Howard Green’s birthday.

To the best of Green’s knowledge, no one had ever gotten human keratinocytes to grow outside the body, in culture, in such a way that permitted them to appreciably proliferate. Biologists had been trying to cultivate skin for a century or more, but no one had hit upon the right method. For that matter, it was difficult to convince almost any brand of cell to divide and grow in a culture dish, and that was because no one was altogether sure of what to feed cells—what culture conditions appealed to them. Once in a petri dish, more often than not cells languished and died, halting any further investigation into their behavior. Fibroblasts and a few other connective tissue cells were among the few exceptions. Less finicky than other cells, they were easily maintained on a standard fare of vitamins and calf serum.

The lure of being able to culture epidermis wasn’t hard to understand. What a fabulous resource surgeons would have if they could take, for instance, a little bit of a burn victim’s skin and multiply it many times over, until there was enough to cover the injured area. To heal, a skin wound needs protective covering. But in the case of many burns, a wound can’t heal because epidermal cells at the wound site are destroyed, and the dermis layer below the hair follicles is unable to reconstitute the epidermis. For over a century, epidermal skin grafts had performed a terrific service in this regard. So remarkably, a person’s epidermis could withstand being detached and reattached to another location on his or her body. The skin graft had to come from the same person (an autograft), because otherwise skin taken from a donor would be rejected by the receiver’s immune system without fail. That left the door open for a major dilemma. If someone was badly burned over a large area, they might not have enough healthy skin left for grafting.

There was a secret to a successful skin graft, and over time that

secret had been revealed to be stem cells. When removing a piece of skin, a surgeon had to make sure to shave off the section deeply enough to include the bottommost layer of the epidermis—its basal cells—along with some underlying connective tissue. Otherwise, when the piece was transplanted, like a sapling that fails to establish its root system, it might not take hold. The basal cell layer was important to include, surgeons had discovered, because it contained stem cells that enable the skin to regenerate. They constantly churn out new cells that progress through the outer layers of the epidermis toward the surface, die, cornify, and are shed, with other cells rising to follow. Human skin completes this bottom-to-top, birth-to-death cycle within three to four weeks.

“At that time, no one in the profession thought in actual terms of stem cells,” explains Green. “But they knew that if they made a good graft, the grafted cells would provide progeny for the life of a person, so that implied stem cells.” In keeping with McCulloch and Till’s definition of a stem cell, basal cells deep down in the epidermis produced lots of progeny and enjoyed long survival. Just like stem cells from bone marrow, they had permanency.

Historians claim that the first person to successfully graft animal tissue was none other than Abraham Trembley, that wonderman of hydra research. An experiment that Trembley undertook in 1742 presented him with the strange sight of the tissues of two hydra growing together, and meanwhile shows us what deft hands and instinct he must have possessed.

Somehow overcoming a hydra’s smallness—most are no longer than one-third of an inch—and drawing upon his knowledge that a hydra is essentially a hollow tube, he began his experiment by turning one of his tiny green creatures inside out like a sock. But this

sleuth’s imagination was only just warming up, for he then strung this inverted hydra onto a boar’s bristle and proceeded to push it into another hydra’s mouth, positioning it all the way down inside this second hydra. A few days later he saw something quite remarkable, which was that inner tissue of the outer animal had fused with outer tissue of the inner animal.

“I cannot explain what became of the body of the inner polyp, whether it was dissolved in the stomach of the outer polyp, or whether the inner body merged with the outer,” he later wrote in his memoir. “I can definitely state, however, that I could still see the body of the inner polyp lying inside the outer polyp several days after it had been inserted. As for the inner polyp’s head, I was certain that it had fused with that of the outer polyp. The lips of the outer polyp adhered to the neck of the inner polyp, and after a certain time the two heads of these polyps formed but one which had two rows of arms.”

Ever since, grafting tissue has imposed a menacing complication. Unless a donor’s tissue is compatible with the recipient, it can easily be rejected, its cells facing attack—in the case of humans—by lymphocytes dispatched by the recipient’s immune system. Trembley didn’t run into this problem in the above experiment, because the two hydra he used were from the same species, their tissues a near match. But had they been from different species, in all likelihood their tissues would not have fused. Hydra don’t have lymphocytes, but “they certainly have some active form of an immune system,” maintains Hans Bode, a biologist at the University of California, Irvine, who has been contentedly studying hydra for over thirty years.

As twentieth-century biologists have come to appreciate, a hydra is replete with active stem cells. Its body column consists of two one-cell-thick layers of epithelial cells—the ectoderm, which lines the animal’s outer surface, and the endoderm, which lines the gut cavity—and the cells of both layers behave like stem cells, according to Hans Bode. As these stem cells do their thing, which includes continuously dividing while sometimes carrying on a function like digestion

(unlike stem cells in more advanced creatures, whose sole function appears to be producing progeny cells), they move outward, not unlike the way stem cells in the basal layer of human skin do. “The geometry is a bit different, but the concept similar,” shares Bode. “There’s a steady state situation in which cells produced in the middle of the animal are displaced toward the extremities. Those in the upper body column are displaced into the head and out to the tip of the tentacles where they are sloughed. Those in the lower body column get displaced into the foot and sloughed at its base.”

Biologists would take up the idea—how could they not?—that a hydra’s wealth of stem cells accounts for its phenomenal ability to regenerate. Its capacity to regrow and remodel itself, they would submit, requires more than just stem cells; other internal forces, for instance, organize the new tissue within the context of the whole. On the other hand, if it weren’t for a hydra’s multitude of stem cells and their tireless yield of new cells, the hydra would be at a loss for new tissue and wouldn’t be the superb regenerator that it is. Notes Bode, given Trembley’s discoveries about hydra, “Trembley’s discovery was regeneration.” Which goes to say that, even though he would never know one, his discovery was the stem cell as well.

In the fall of 1974, Green and Rheinwald were unknowingly poised to extend the practice of grafting human skin beyond its current limitations.

Once they had put cells they knew to be foreskin keratinocytes into a dish with 3T3 fibroblast feeder cells, they didn’t have to hold their breath for long. After a few days they observed that their keratinocytes, nourished by the 3T3s, were growing “amazingly well,” describes Green. “They grew better than we hoped.” Now all was revealed. The mystery cell that had stolen their attention just

happened to be the special cell that serves as the regenerative basis for human skin, and the 3T3s they had used to support its growth happened to be the perfect companion, for in the real life setting fibroblast cells in the connective tissue that underlies the basal layer stimulate the proliferation of its cells. Without the 3T3s, their skin keratinocytes “wouldn’t have grown at all!” notes Green.

To suddenly realize that they had crossed River Impossible and had skin cells flourishing in the foreign environment of a dish—the surprise couldn’t have been greater. “It was a happy event,” says Green thriftily. A happy accident as well, he acknowledges. Here people had been attempting to cultivate skin cells for ages, and he and Rheinwald had done it without any intention of doing so.

The MIT biologists would find that as long as they used their 3T3 fibroblasts as nursemaids, they could grow keratinocytes not only of the skin, but those found in every other squamous epithelium tissue. Pretty soon they were propagating keratinocytes taken from the human cornea, the pharynx, the vagina, the esophagus, and all regions of the mouth. Their laboratory hummed with incubators filled with this cell—a type of human cell, remember, that never before had been made to proliferate outside the body to any great extent.

Keratinocytes from skin remained the researchers’ main interest. Certain proteins known as “growth factors,” they found, quickened the cells’ proliferation rate. Still, they had trouble keeping the cells multiplying, because they tended to differentiate into mature cells and stratify. Bit by bit, however, the investigators devised ways of improving their culturing technique. By transferring the cells to fresh flasks more frequently, and applying an even more judicious choice of growth factors, they were able to maintain most cells in a proliferative state, which resulted in enormous numbers of cell progeny in a surprisingly short time.

So successful were they in fine-tuning their methods, Green and Rheinwald were able to expand a piece of epidermis the size of a postage stamp to more than 5,000 times its original size. This was

roughly the surface area of an entire adult human being, or about twenty-one square feet. Growing up this quantity of cells took approximately three weeks, the numbers of keratinocytes in culture doubling every seventeen hours. As Green observed, the culture conditions that his lab had worked hard to optimize enabled epidermal cells to grow faster than they did in the body.

It became increasingly clear that the essence of the scientists’ culture method was its preservation of keratinocytes that had a strong degree of stemness. The procedure of transferring cells to new flasks in a timely fashion perpetuated small keratinocytes that were as strikingly proliferative as the large cells in their culture were unproliferative, and the scientists were all the more certain that the small cells they were urging to grow were equal in behavior to the stem cells in the basal layer of the epidermis that perpetuate skin renewal. “We knew that the small cells in our flasks were proliferative—that they were basal cells,” notes Rheinwald, who is currently the associate director of the Harvard Skin Disease Research Center at Brigham and Women’s Hospital.

Today, these regenerative skin dwellers are regarded as either adult stem cells or their more differentiated kin—progenitor cells. They are less flexible than an embryo’s stem cells, but they have enough stemness to produce vast numbers of epidermal progeny. A recent finding raises the possibility that epidermal keratinocytes could possibly harbor a latent ability to produce other types of cells. As shown by Michele De Luca and Graziella Pellegrini in Rome, keratinocytes that form the eye’s conjunctiva, the membrane that lines the eyelid, give rise not only to other keratinocytes but also to the conjunctiva’s mucus-secreting globet cell. Previously, these two ocular cell types were thought to originate through separate lineages.

Howard Green couldn’t help but realize what his lab’s revelation might mean for surgeons who were in constant need of epidermal cells, whether for the sake of covering burns, or replacing ulcerated or scarred skin, or skin ravaged by a blistering disease. “We immediately knew that we had something that would make possible the

cultivation of this cell type in a way far superior to anything that had existed before,” Green recounts. But if push came to shove, could their flask-grown epidermis be converted to a useful form of therapy? With patients in mind, he set about designing the following protocol:

• grow up the keratinocytes

• disassociate them with an enzyme and transfer them to new flasks

• grow the cells up again and detach the entire sheet of cells from the bottom of the glassware with another enzyme

• wash the sheet to get rid of any traces of enzyme

• gingerly staple the sheet to a thin petrolatum gauze

• place the skin with its gauze backing onto a prepared wound and suture in place

• remove the gauze a week or so later

Green first applied this skin-growing technique to a living animal in 1980. After preparing a sheet of cultured human keratinocytes, he and a postdoc used it to cover a small wound on the back of a “nude” mouse, handling the tissue ever so carefully since it was as thin and friable as wet Kleenex. The mouse came from a strain that not only was furless, but lacked an effective immune system. Thus it wouldn’t reject the human graft, which in the weeks ahead took hold nicely. The mouse’s skin, once healed, was seen to be made up of both the graft’s human epidermis as well as mouse epidermis, which had grown in from areas bordering the grafts.

Not long after that, Green tried his new technology on people. He and a colleague prepared cultures and clipped them to gauze for Dr. Nicholas O’Connor, a prominent burn surgeon at the Peter Bent Brigham Hospital, who sutured these epidermal sheets onto the small burns of several patients. Nearly half of the cultures engrafted and contributed to the skin’s complete recovery.

But Green’s culturing strategy would receive a far, far greater test

in 1983. One day Green, who had moved on to Harvard Medical School, received a call from John Remensynder, a surgeon at the Shriners Burns Institute. Three young boys, Remensynder related, had sustained severe burns over more than ninety-five percent of their bodies. They had been painting, and as they cleaned themselves off with a solvent, one of them had struck a match to light a cigarette. One boy had died soon after the accident. The other two, brothers ages five and six years old, were expected to expire as well, since as a rule children burned that badly couldn’t survive. Dressings that included strips of cadaver skin and sheets of collagen would temporarily assuage their wounds, but sooner or later their bodies would reject these foreign materials. What both boys needed for engraftment, but didn’t have enough to spare, was their own healthy skin. Remensynder wondered if Green would be willing to lend a helping hand by providing the burn surgeons in charge—Nicholas O’Connor and Gregory Gallico—with his innovative skin-growing strategem.

“We were a basic research lab, not a hospital lab, and were poorly equipped to do anything on that scale,” recalls Green. “It would take lots of cultures and lots of people to prepare the cultures and transfer them from our lab on Longwood Avenue across town to Shriners.” Yet he knew full well that if the attempt wasn’t made, the boys didn’t stand a chance.

And so Green, his five postdoctoral fellows, and two technicians rolled into action. As it would turn out, they would culture 146 grafts for one brother, and as many as 233 grafts for the other brother, whose wounds proved more problematic. Each graft measured approximately seven square inches. When early-morning surgery was called for, Green and his team would arrive in the lab by 5 a.m. in order to give themselves enough time to staple the specimens onto a gauze backing for easier handling, set them back into petri dishes, package the dishes in boxes, gas and seal the boxes to ensure germ-free air quality, and oversee the boxes’ transport to Shriners. The technicians and postdocs in his lab rose to the challenge with a gusto

he’ll never forget, Green proudly relates. At the same time, there’s every indication that Green himself proved an inspiring leader. Carolyn Compton, a pathologist now at McGill University who was part of the Shriners team that worked on the two brothers, recalls being struck by how “calmly and fearlessly” Green pressed forward in his endeavor to take a new concept straight to the patient. “At first, he really didn’t know what he had,” relates Compton. “But the minute he learned how to culture these cells and expand them in vitro, he played the iconoclast and was ready to try to apply them in the clinical setting. A couple of months with mice—and boom, right into patients.”

In the early weeks, the children’s red and raw bodies made for such a gruesome sight that Green sometimes wondered if he were doing the right thing. Wouldn’t it be more merciful to let the boys die? But a nurse at Shriners said something that stopped his nagging doubts. Recounts Green, “She told me how one of the boys, just before receiving general anesthesia in the operating room, had pleaded with her not to let him die. As burned as they were, these children had a fierce desire to live, and so I became fully reconciled with trying to help them.”

The Shriners staff would witness what amounted to a miracle. Small patches of skin salvaged from the armpits of each boy and expanded 10,000-fold in Green’s lab meant the difference between life and death for each brother. In essence, the stem cells in those remaining underarm patches of epidermis saved their lives. Due to its resplendent regenerative capacity, this cell source would serve to replace fifty to sixty percent of each boy’s total skin surface. Another source of stem cell would enter in as well. In skin areas that had escaped with second-degree as opposed to third-degree burns, stem cells that live deep in the hair follicle survived and were able to return those areas of skin to near normal.

Within a few weeks, both boys had achieved medical history simply by being alive. At first their thin grafts were delicate and vulnerable, but in the ensuing months the new epidermis became

more securely anchored to the underlying connective tissue. It would require another two to five years, and yet the connective tissue beneath the restored epidermis would also heal and return to normal. “It was the first demonstration that human stem cells of any type could be expanded substantially in culture and used to permanently restore a patient’s lost tissue,” says Rheinwald. With a bone marrow transplant, it wasn’t necessary to multiply the marrow’s blood-forming stem cell, because the body saw to that. But in the case of widespread skin loss, growing up supplemental skin cells was necessary.

Having shown that real skin can be custom-grown in the lab, Green would soon be hailed as “the father of skin culture.” Many in medicine would make note of this biologist who, working “far ahead of the curve,” in the words of Pamela Robey, chief of the NIH’s Craniofacial and Skeletal Diseases branch, was “developing techniques for tissue regeneration well before it was fashionable.” Here, taking its place beside a bone marrow transplant, was another outstanding example of regenerative medicine that allowed for treating the body with its own cells. One distinguishing mark of Green’s work was the way it so smartly overcame the main reason so few cell-based treatments yet existed, which Green today succinctly sums up as “a culture problem.” Harvesting cells from the body, keeping them alive in culture, and expanding them constituted one of the tallest orders that regenerative-medicine scientists would face.

Green made his new skin-growing technique available to patients everywhere by forming the company BioSurface Technology, which in 1994 was purchased by the larger biotechnology firm Genzyme in Cambridge, Massachusetts. Genzyme’s product Epicel continues to be sold and at present is the only commercial skin-replacement product of its kind for large-area burns. The harsh reality is that because so few people who are badly burned survive, Genzyme receives less than a hundred requests for the procedure each year, according to a Genzyme spokesman. With smaller burns, the kind most often needing treatment, the primary skin-replacement method continues to be skin grafting, according to Carolyn

Compton. “The skin graft is much too useful, too reliable, and too cheap to ever do without,” she cites.

Green’s method of growing basal-layer keratinocytes and crafting them into transplantable sheets of skin, while spectacularly innovative, has its weaknesses, Green himself admits. It is labor-intensive, given that it takes several weeks to provide a patient with skin. Plus the resulting sheet—all of two to three cells thick—is fragile and easily torn.

What comes as no surprise is that over the last several years many other researchers have tried to culture durable skin using both natural and synthetic materials. Surgeons would love not to have to rely on a patient’s own tissue and instead have plenty of skin on hand for repairing skin. According to various authorities, some of the resulting formulas contain proliferating basal keratinocytes, which makes them therapeutically useful, whereas other formulas have proven inferior, their makers failing to base their concoctions on skin cells that have an adequate degree of stemness.

A major stumbling block, says Pamela Robey, is that there is still no easy way to select for epidermal stem cells, maintain them, and increase their numbers without some of them maturing and losing stemness. “And if you try repopulating a wound with engrafted material that doesn’t contain stem cells, the effect is not going to be very enduring,” she offers.

In the meantime, Green and Rheinwald’s demonstration that keratinocytes of the cornea, esophagus, mouth, and genitals also could be significantly multiplied outside the body would spark the interest of other researchers, who are currently investigating a range of related medical applications. Here again, the secret of the culture lies in promoting the growth of only those keratinocytes imbued with stemness. Among the transplant procedures being worked on is a method for replacing mouth-situated epithelium that has been reduced by gum disease and mending tissue in the middle ear that has been lost to infection.

When last heard from, the two brothers whose lives were saved

at Shriners Burns Institute three decades ago were still living close to their birthplace in the Midwest. One brother reportedly was doing fine, whereas the brother whose skin replacement had previously posed problems was said to have encountered related health issues. The long-term effectiveness of cultured epidermis is by no means a given, with many an associated complication unresolved. Howard Green, meanwhile, still marvels over the fact that a tumor in the testicle of a mouse put him on the road to culturing skin, whereupon he was able to offer this precious material to others.