From Jones and Bartlett, a book on Stem Cells from Dr. Ann A. Kiessling and Scott C. Anderson:

Selected Articles:

April 16, 2004

A Baby's Hair

The first animal cloning was performed with a baby's hair and tweezers.

 "And what a glorious society we would have if men and women would regulate their affairs, as do the millions of cells in the developing embryo." -- Hans Spemann, 1938

Hans Spemann was in a foul mood as he rearranged his blankets. The beginning of the twentieth century should be vastly more exciting than this, he thought. Being swaddled in a lounge chair on the sanatorium porch was not his idea of a glorious way to ring in the new century. Getting tuberculosis was damned inconvenient, and the recovery was almost as bad as the disease. He hoped the book he had just bought would keep him from going comatose.

He anxiously thumbed through the first pages of The Germ Plasm: a Theory of Heredity by August Weismann, the famous zoology professor. It was not light reading. Nevertheless, as a recently graduated biologist, Spemann found it to be a great tonic.

Spemann realized that biology was undergoing a renaissance after centuries of neglect. Even hoary old Aristotle was being dusted off. Like all good biologists, he knew of Aristotle's works, and it was unbelievable to him that classical science could have been eclipsed by magical thinking for almost two millennia.

Incredibly, Aristotle's chickens still represented the state of the art in embryology. But this long dry spell was about to end. Spemann couldn't know it yet, but he would be one of the scientists to open the floodgates.

Spemann was aware of genetic material and how it governed heredity from Mendel's recently rediscovered work with peas. At the monastery in Moravia where he studied, Mendel had shown that plants inherited characteristics from their parents in a curiously mathematical fashion.

Mendel had studied to be a monk, but fortunately for science he flunked his teaching exams and reluctantly retired his robes. As a layman, Mendel was no longer bound by the religious strictures against biology, so the monastery was able to authorize his famous pea studies. It was a remarkable example of the Church reaching out - albeit with a heavily gloved hand - for some of the tempting fruits of biology.

Since the dawn of civilization, philosophers and religious scholars have created fanciful stories to explain life. But on a parallel track, society was also stockpiling secular information. Farmers fared better as they learned more about plants and animals, and that hard-won information was passed down through the generations. As time went on, the religious mythology adapted to better conform with that accumulating knowledge. It was not always an easy fit. Fluids and gases with magical properties called humors were invoked - with a lot of hand-waving - to ascribe function to the vessels and fibers that were undeniably a part of animal tissue. Angels were often summoned to patch up the particularly difficult parts.

It was a kind of biology, but certainly none too rigorous. The eighteenth and nineteenth centuries saw an improvement, but it wasn't easy to recover from the intellectual torpor of the Middle Ages.

Early science involved more observation than experimentation. The concept of interfering with normalcy in order to know it better was neither intuitive nor popular. But as the power - and the demands - of science became better understood, great strides were made. Careful observations, open inquiry, repeatability - these important touchstones spread throughout the world as science tentatively took root again. Life spans again started to rise and the quality of life improved in direct proportion to the application of the scientific method.

Still, when Mendel published his paper in 1866, it immediately sank into oblivion. It would take thirty years for the world to appreciate the magnitude of the failed Monk's work. Rediscovered at the turn of the century, Mendel's pea studies - along with Weismann's book - represented the latest thinking about heredity.

Spemann was impressed by Weismann's theorizing. He couldn't help feeling though, that Weismann needed more research to back him up. There were a lot of leaps of faith between the scanty data and his important pronouncements.

The central problem is easy to state: animals grow from a single fertilized cell, yet they are ultimately composed of hundreds of different cell types. How can you explain this profusion of cell types? How do they manage to assemble themselves into organs and how do the organs know where to go? Phrased less scientifically, where do we come from and how did we get here?

Weismann theorized that when a cell splits, the genes are chopped in half. Each new cell gets a different half. Imagine that the first cell has genes corresponding to four cells, a heart cell, a neuron, a liver cell and a gut cell. After the first split, the two resulting cells - called daughter cells - would have different genes. One cell might have genes for a heart and liver, the other for a brain and gut. After each of these daughter cells split, you would have four cells, one for each organ.

Of course animals have more than four organs, so you would need to start with more genes, but in general, Weismann thought, each new generation of cells would have fewer and fewer genes, until just one gene was left - and that final gene would presumably determine what kind of a cell it had become. It was a thoughtful hypothesis. And, with any luck, it was testable.

Spemann knew that Weismann's theory was similar to that of Wilhelm Roux, who called it "qualitative division," since cells change their genes every time they divide. Roux had actually conducted an experiment in 1888 that seemed to validate the theory. Using a hot needle, he killed one of the cells in a two-cell frog embryo. The frog that resulted from the surviving egg was deformed and Roux claimed that he had created half a frog. It seemed pretty convincing, but it was also remarkable because it was a bona-fide experiment, one of the few since Strato carried out his experiments at the Lyceum, over two thousand years before.

Roux felt that his results vindicated the theory of preformation. If the frog embryo is just a miniature of the adult and you cut it in half, you should expect to get half a frog.

But a scant four years later, Hans Driesch seemed to come to a different conclusion. By simply shaking them, he separated the cells of a sea-urchin embryo at the two- and four-cell stage. He was surprised to see that each of these cells were capable of generating an entire urchin, although they were smaller than normal. Driesch convinced himself (if few others) that these results implied the existence of vital spirits. However, he was pretty much the last biologist to think in those terms.

Did these experimental differences hinge on the fact that urchins are far simpler than frogs? Or were the scientists simply bad at the art of science? It was hard to reconcile the data, but it seemed preposterous that you could get whole creatures from embryo bits.

An experiment started to take shape in Spemann's fevered brain. It might be tricky, but it would definitively prove or disprove Weismann's theory and settle the debate between Driesch and Roux. But first, no matter how frail his health, he would have to leave the sanatorium. After giving the Alps a final salute, he packed his bags and headed for Wurzburg, a quaint city on the Main river.

Spemann found work at the University of Wurzburg, where he lectured while he worked toward his professorship. Finances were tight, and Spemann and his wife elevated frugality to a high art. The impending birth of a child laid a fine veneer of anticipation over their lives. But the intellectual tone of the times allowed his imagination to soar, and overall, he was happy.

His experiment would require an incredibly delicate manipulation of eggs. Spemann was becoming well known for his prowess with microsurgery, but this would test even his skills. He chose salamander eggs, and for good reasons: they're large, easy to incubate, transparent and readily available.

Spemann was a master craftsman and created some of the finest micro tools in all of Europe. Deftly twirling glass rods over the Bunsen burner, he would soften and then carefully stretch them into silk-thin strands. These he would use as micro-fingers to manipulate the slippery salamander cells. His dexterity with these tiny tools, he had to admit, was a major factor in his attraction to biology. Mastery counts. For the task he had in mind, he would need his finest probes.

But salamander eggs are sticky; they can't simply be shaken apart like sea urchin eggs. After many failed attempts, he was ready to abandon the entire pursuit. His daughter Margrette had just been born, and the seriousness of raising a family was sinking in. If he couldn't get this experiment to work, he would have to return to his old job at his father's publishing house.

Spemann paced around his tiny living room. He held his daughter, patting her back and thinking. What he needed was a new kind of instrument. Not just a finger-like probe, but a flexible and thin lasso. If he couldn't pick the embryo apart, he might at least be able to tie it off. But no matter how thin he drew the rods, whenever he used one of his glass threads as a ligature, it quickly snapped apart.

As little Margrette gurgled and burped milk down his shirt, an idea began to take form. Would a hair work? Not an adult hair, which he knew was too thick and brittle. But ... he looked at the top of his daughter's fuzzy head.

He sat the child down, grabbed a scissors and snipped a wispy lock of hair. Thus armed, he headed off to his lab. From his collection of fertilized salamander eggs, he selected one that had just cleaved in two. If he could separate these two cells, what would happen?

Carefully, he used tweezers to tie the thin hair around the budding embryo. Salamander embryos have a jelly-like membrane around them for protection, and Spemann tied his loop right around the middle of this membrane. He carefully tugged on the two ends of the hair, squeezing the two freshly divided cells into opposite halves of the egg's membrane. He pulled tight to make sure that they were separated, but not so tight as to rupture the membrane.

If Weismann and Roux were right, each of these two cells should grow into something different - but what? Perhaps one would become the head and the other would become the tail. Or maybe one would be the back side and the other the belly. But now, cleaved by the hair lasso, would these parts even be able to continue developing?

It only took a few days for the embryo to grow, but it seemed to take forever. Finally Spemann pulled a chair up to the bench, and placed the embryo under the scope. What he saw made him rub his eyes in disbelief - there in the dish were two identical salamander embryos, both of them whole and normal.

Figure 1. Recipe for twins: loop a baby hair, position it around a cleaved egg, then tighten.

Spemann called it twinning, but we know it today by a different, more loaded, name: Spemann had documented the first in-vitro animal clone, and it was more than a little perplexing.

Biologists often think about the "machinery" of life, especially when it comes to embryology. The egg is a wondrous, one-of-a-kind machine that creates more machines with each generation. But what kind of machine could be cut in two and still retain its functionality? How could half of a machine go on to make two fresh machines?

Spemann's result directly contradicted Weismann. At least at this point in development, the genetic information was certainly not splitting apart, since both cells contained all the information needed to create an entire salamander. This was a major crack in the theory of preformation.

Not all of Spemann's lassoed embryos yielded twins. Some resulted in a single embryo with a strange-looking "belly piece" attached to it. A good scientist knows how to milk the most from an experiment, which is often tedious, expensive, frustrating, lengthy and sometimes - as is the case with amphibians - seasonal. Spemann didn't throw out these "failures." Instead he made an inspired conjecture that success hinged on how the egg first split.

A fertilized salamander egg is not without structure. A gray crescent can be seen to form after fertilization at one end of the egg. When the egg first cleaves, it has two simple choices: it can either split that crescent in two or not. If the egg split so that a piece of the crescent was in both daughter cells, Spemann got twin salamanders. It was in the odd instances where the egg cleaved into unequal daughters, one with the crescent and one without, that he got the belly pieces.

Two concepts were coming into focus. First, wherever and whatever the genes were, when they split they still seemed to contain enough information to make two complete individuals. Second, the cytoplasm somehow carried information, too. Without the cooperation of both, you couldn't guarantee twinning. The belly piece was the occasional result of an egg that happened to divide unevenly: the daughter cell with the gray crescent grew into a whole salamander, but the other cell, lacking the right stuff, just formed a blob of belly tissue.

Spemann was thrilled; he had found a reliable way to test the great professor's theories. In the following months, Spemann tried hundreds of variations on this experiment and showed that he could create individual salamanders by tying off the embryo at the two, four and eight-cell stages. After the eight-cell stage the embryos either failed to form or formed improperly. He decided that something in the cells must be changing at that point - their fates had been determined - so he called this process determination.

Today we know that these primitive cells are able to give rise to whole animals because they are embryonic stem cells. They have the potential to become any type of cell, and it is not until later in development that they declare their special destiny.

Figure 2. Cloning from an older cell. After tying the embryo, the side with the nucleus grows. Then, at the sixteen-cell stage, a mature nucleus is pulled back, creating another salamander.

But Spemann wanted more evidence. Was the stuff of genes contained in the cell fluid or the dark nucleus floating in the center? When, exactly, did the cells become determined? Could a different nucleus be transplanted into an egg? Would it still grow properly? Incredibly, Spemann thought up a single experiment that might answer all these questions at once. All he needed was a little more hair.

This time Spemann used the precious baby hair to tie up a fertilized egg immediately before the first cleavage. He wrapped a noose around the egg and carefully tightened it around the middle, forcing the dark nucleus into just one half of the cell.

It was a difficult experiment, but one of the dumbbell-shaped eggs flourished. He watched, hunched over the microscope, as an embryo formed on the side of the egg with the nucleus. The other side, containing clear cellular fluid, showed no growth. Apparently, the genes were in the nucleus. That wasn't a total surprise to the biological community, but it was a good confirmation.

This was only the first stage of the experiment, though. Following several divisions of the embryo, Spemann carefully loosened the loop of hair. After a few minutes, a nucleus from the embryo slipped into the clear part of the egg.

Presumably, this nucleus had divided along with the other cells in the embryo, so it was more "adult." Quickly, Spemann tightened the loop again, trapping the wayward nucleus and separating it from the rest of the embryo.

Spemann had created a new cell made up of the original egg fluid and a new, more mature, nucleus. He watched it intently, checking every few minutes to see what would happen to this small pocket of protoplasm. To his excitement, it went on to form a second embryo. Again, it seemed that - at least for the first few divisions - the nucleus of a daughter cell was still capable of forming a whole new embryo. If the genes were being segregated by cell type, it clearly wasn't in the first few divisions. How could a mere gene fragment have the power to create a whole new animal? If the genes were split at all, it must occur later in development.

It was a simple and elegant experiment. Spemann had invented cloning by nuclear transfer. He had moved the original nucleus out of an egg and then put a more developed nucleus back in its place. It was a very neat trick, and it was all accomplished with two tweezers and a thin strand of baby hair.

Figure 3. Some of the first microsurgery tools created by Hans Spemann and Hilde Pröscholdt.

When Hilde Mangold came to work in his lab, Spemann immediately recognized that her skills at the microscope exceeded his. Hilde was starting a family and an interesting new experiment at the same time. The new research involved transplanting tissue from a special spot on a later-stage embryo.

For the first few hours of their lives the tiny salamander embryos are basically smooth, hollow spheres. But after a while, features start to emerge. One of the earliest features looks like nothing more than a little dimple, called the blastopore. This was the spot they decided to use in their new transplant experiments. One day, Hilde took a slice from this dimple and transplanted it to a smooth part of another embryo.

To everyone's surprise, it wasn't a leg or a tail that popped out, but most of a whole new embryo. Nerve cells, backbone, head, eyes - they were all there, and the entire team was baffled. As with the other experiments, this new salamander was the color of the host embryo, which apparently had been induced by the graft to form a whole new body.

Powerful stuff, to be sure. The blastopore tissue, they decided, must be very special. It seemed to have all the information needed to organize the creation of a whole animal, so they called it the organizer.

Figure 4. Transplanting a bit of tissue from the "organizer" can create two-headed salamanders or even twins.

Hans Spemann and Hilde Mangold wrote up their paper - and electrified the biology community. Scientists around the world duplicated and verified their results. More questions were posed and more experiments were performed to answer them. It seemed like it should only be a few more years before the inducers themselves would be isolated, and embryology would be an open book.

Two years after her marriage, Hilde gave birth to a son, Christian. He was quickly enlisted as a hair donor, and the work carried on. Hilde hustled back and forth between the lab and the small apartment she shared with Otto, happy to be involved in such exciting work.

One particularly hectic day, in between transplants, Hilde found herself preparing yet another bottle of milk for Christian. She walked into the kitchen to heat up the milk, and struck a match to light the little gasoline stove.

An explosion shook the building. After the plaster quit falling and the dust had settled, Hilde Mangold's lifeless shape could just be made out, sprawled on the kitchen floor. She would never know how pivotal her research was, and how famous it would become.

The paper she wrote with Spemann attracted the attention of the Nobel committee. The Nobel prize is never granted posthumously, so although it was her work that was being honored, Hilde herself was not named. Hans Spemann accepted the award by himself in 1935. In recognition of her work, he always referred respectfully to "The Experiment of Hilde Mangold" in his published papers.

Although he tried repeatedly, Spemann was never able to discover or isolate the inducer. It would take another seventy-five years to finally discover the vanishingly small quantities of chemicals that were coercing the cells to change their fate.

Spemann went on to propose a "fantastical experiment" - the transfer of an adult nucleus into an egg cell. This, he felt certain, would be a way to clone a human being or at least human tissue. That, in turn, could possibly be used to cure diseases.

The Catholic Church was horrified, claiming that research like his was directed toward two goals: the destruction of all mankind, and the destruction of god. Inevitably, unflattering comparisons to Dr. Frankenstein were made. Spemann's experiments, with his patchwork salamanders, seemed a little too close to Mary Shelley's nightmarish vision for some people.

Spemann's contributions to biology may have been shaded by the pervasive philosophy of the time. Nationalism was on the rise as Germany painfully rebuilt after the First World War. A fascination with a strong, paternalistic state was infecting the German zeitgeist. Nazism and Communism were the revolutionary politics of the time. Both posited that a powerful central government could do much more than a loose federation of citizens.

How could it be otherwise? Surely with the smart people running the show, executing proper five-year plans, the world would be much more efficient. In this environment, it was easy for Spemann to attribute a top-down structure to the embryo, as he did with the organizer. It wouldn't be the first time that politics had insinuated itself into scientific research and, probably, it wouldn't be the last.

Ultimately, however, there is something philosophically unsatisfying about the organizer. It's reminiscent of the old-fashioned ideas of pre-formation that Aristotle had so effectively rebutted two thousand years before. If the blueprint for the embryo comes from the organizer, where does the organizer come from? Has anything really been explained, or is the mystery just pushed farther back?

This is the second installment of a series on the history of cloning and stem cells. The first was "Aristotle's Chickens." Interested in more? Drop me a line!

Copyright © 2000-2014 by Scott Anderson
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Here are some other suggested readings about cloning: