Earlier this month, President Bush voiced strong opposition to use of human embryonic stem cells either for reproductive or therapeutic purposes. The U.S. House of Representatives has legislation to enforce this double negative. The Senate is thought unlikely to take action on the cloning issue. (See BioWorld Today, April 11, 2002, p. 1.)
Meanwhile, scientists in many countries are cloning an ever-widening assortment of animal life, with various objectives, including eventually human cell and tissue replacement. Among them is British developmental biologist John Gurdon, at the University of Cambridge in the UK. He followed a hunch that what others thought were doomed cloned embryos might still yield a source of stem cells. “Even the highest cloning efficiencies in mammals,” he pointed out, “have not exceeded 6 percent of total transfers yielding live births. The 94 percent or more that do not undergo nuclear reprogramming,” he added, “become abnormal and die.”
Gurdon is senior author of a paper in the Proceedings of the National Academy of Sciences (PNAS), dated April 30, 2002, but released online April 23. Its title: “From intestine to muscle: Nuclear reprogramming through defective cloned embryos.”
“What our work shows,” Gurdon told BioWorld Today, “is that we can retrieve healthy cells able to grow indefinitely from embryos, which themselves cannot survive more than a day. When one does cloning experiments,” he pointed out, “that means nuclear transplantation from specialized tissues. Most of the embryos thus obtained develop very abnormally.
“In our experiments with frog embryos, we found that only about 1 percent of such early embryos actually developed normally. All the rest died very abnormal.
“What we did,” Gurdon continued, “was to take cells from these abnormal early embryos because they had a few normal cells as well as much dead tissue. Then we grafted those cells onto a normal host embryo. Surprisingly, the derivatives of those cloned cells, which in this case came from the intestine, nevertheless proved capable of making quite a range of other cell types.
“We happened to concentrate on muscle, and perhaps most surprisingly, they went on growing probably indefinitely at least 100 days or so. That outcome was not expected,” he went on, “because the embryos, in our case, were destined to die within a day.”
Embryos Get Green Light To Convert Gut Cells
Gurdon recounted his three-step cloning procedure. “First, we took a piece of functional frog tissue in our case intestine epithelium removed a cell nucleus and put it into unfertilized eggs, whose own nucleus had been removed. So the embryos that developed came from the nucleus of what was once an intestinal cell. The starting material the intestinal cells were marked with green fluorescent protein [GFP], so we could follow their fate through the cloning process.
“Our second step was to treat these cells with transforming growth-factor-beta [TGF-beta], which guides these now-embryonic cells into a particular path of stem cell differentiation. Growth factors are gene products, normally acting throughout life to tell cells what to become. And they are available as purified proteins. When we added TGF-beta to those embryonic cells, it was very effective in telling them which way to differentiate. We grafted them into the region of the embryo from which muscle normally comes. That was the second step for directing these cells. In our particular case, we chose body muscles as the direction to send them in.
“We had by then rejuvenated the intestinal cells back to an embryonic cell by the nuclear transfer procedure. Next we treated them with a particular concentration of TGF-beta molecule, which set them off in the direction of forming mesodermal or muscle tissue derivatives. We followed the green fluorescent-marked cells, which are the intestine-derived ones, and found that those survived very well, making muscle, notochord [backbone precursor] epidermis and other frog tissues. Perhaps most remarkably, they continued to grow to the end of the experiment about 100 days.
“This all went on in the African clawed frog, Xenopus laevis,” Gurdon remarked, “which are very favorable models easier than mice for this kind of embryological experiment.”
Tadpole Tail Muscle Stops Short Of Frog
The stage of amphibian metamorphosis was the climax of the team’s tissue-differentiation experiment.
“We used the word metamorphosis,” Gurdon explained, “because that is when a tadpole changes into a frog. And the reason it became relevant to our own experiment was that we chose to demonstrate the derivation of muscle cells from intestine by reference to cells that are predominantly tadpole tail. It uses those muscles to swim around. When the frog undergoes metamorphosis, it loses the tail. So our experiment had to stop at that point. Since the cells we had produced by this means were in the tail, they were resorbed normally before the tadpole went on and turned into a frog. So we couldn’t follow them any more.”
Gurdon sees two major implications in his cloning experiments.
“Similar results very likely would be obtained if the research had been carried out in mammals or even humans,” he observed. “For the moment, we’re not allowed to do that kind of experiment on humans. And you would get a lot of those failures when doing cloning experiments. So in my view, such very early embryos cannot possibly become human beings because they are destined to die in a day or two. We humans have to live longer than that.
“Which means,” Gurdon went on, “if you do these experiments, you should be able to derive these rejuvenated cells from embryos, then use them for experimental work like finding out what makes human cells grow and differentiate so well as a potential basis for embryonic stem cells. Potentially, then they could they be used not to reproduce human clones but perhaps far in the future for therapeutic purposes such as cell replacement.
“Our work is not at all related to reproductive cloning,” Gurdon noted. “It’s inappropriate for that. But it would be useful, in my view, both for research in the short term to find out what makes human cells grow well and differentiate so well, and in the longer term,” he concluded, “as a possible source of stem cell replacement, bypassing the need for using normal human embryos.”