“When Dolly the cloned sheep was published in 1997,” observed embryologist and molecular biologist Rudolph Jaenisch, “it right away raised an important question: Was she really derived from a mammary-gland cell nucleus, poised with lacteal genes to cause milk production? Or did Dolly arise from a somatic stem cell nucleus, which you cannot define? Nobody had a genetic marker to unambiguously identify the nucleus that gave rise to that celebrated cloned animal.
“Most adult cells don’t have stable genetic markers, which can be tracked from donor nucleus to resulting clone,” Jaenisch pointed out. “Immune B and T cells are exceptions. When they mature, their immunoglobulin and T-cell receptor genes are permanently shuffled, so they can produce the unique specificities to fight pathogens.” Jaenisch, who heads a laboratory at the Whitehead Institute for Biomedical Research in Cambridge, Mass., may have laid to rest this burning issue: Can mature terminally differentiated cells be reprogrammed to generate a cloned animal?
He is senior author of a paper in the current Nature, dated Feb. 10, 2002, titled, “Monoclonal mice generated by nuclear transfer from mature B and T donor cells.”
“This finding,” Jaenisch told BioWorld Today, “tells us something about the genome of adult cells which are difficult to clone. This is important to know, if adult cells are going to be cloned for personalized cell therapy. Therapeutic cloning involves replacing the nucleus of an oocyte with the nucleus from an adult donor cell. In our hands,” he went on, “this preimplantation ball of cells growing in culture gave rise to embryonic stem cells [ESCs] that genetically matched the donor, and had the potential to become any tissue in the body. In the future, these ES cells may be used to treat diseases, such as Parkinson’s, diabetes or spinal cord injury, without the complications of organ rejection.”
The Nature paper describes a novel departure from conventional cloning techniques, which Jaenisch calls “the two-step method.”
“We used a genetic marker a rearrangement of immunoglobulin genes, which occur during maturation of B and T lymphocytes in the lymph nodes,” he recounted. “When B and T cells mature, they have to rearrange several genes, which are expressed to make the right immunoglobulins. Then they go into the peripheral blood and wait for an antigen.”
Cloning From Mature Cell: A First
“By cloning an animal’s B and T cells,” Jaenisch continued, “we showed for the first time unambiguous, unequivocal proof that one can indeed clone from a mature, terminally differentiated cell. We took peripheral lymph nodes, which contain about 95 percent B and T cells plus 5 percent non-lymphatic immune cells, such as macrophages. We knew this would be extremely inefficient because people have tried it in the past and failed.
“Therefore,” Jaenisch went on, “we developed a modified two-step approach. Normally when you clone, you transfer the nucleus into an oocyte to develop an embryo, which you then transplant into a foster mother. We didn’t do this. Instead of transplanting the embryos directly to foster mothers, we put them in petri dishes and derived embryonic stem cells. Then in the second step we derived mice from those embryonic stems. This was very inefficient by itself, because we got 1,000 transplantations and only two embryonic stem cell lines. Sure enough, one was from a B cell, one from a T cell.
“These had all the characteristic gene rearrangements. So it was unambiguous proof that these embryonic stem cell lines were derived from B and T cells. We made mice from those ESCs in many attempts. They had gene rearrangements in every bodily tissue, not just in their thymus and spleen. So they were truly monoclonal mice.”
Jaenisch raised the point that “many of the clones people have derived did not arise from terminally differentiated cells but rather from some adult stem cells that are in the population. This hasn’t been tested,” he added, “so we’re testing it now. What we now know is that taking the nucleus from an embryonic stem cell and cloning a mouse is much more efficient than taking a nucleus from somatic cells say, a fibroblast. This is consistent with the idea that maybe adult stem cells, as well as embryonic stem cells, are easier to reprogram than terminally differentiated cells.
“One thing this experiment really shows is that we could make these ESCs, which couldn’t be distinguished from the ES cells obtained from a fertilized embryo. They had full potential to differentiate in every tissue of the body. So we can certainly think now about therapeutic cloning.” Jaenisch cited a thought experiment: “If you make an embryonic stem cell from a patient to derive, let’s say, nerve cells for treating Parkinson’s disease if that becomes feasible or human diabetes, then you would try to get a matched donor to prevent problems of rejection. In any transplantation, if you clone the nucleus from the patient’s own skin, for example, you make an ESC, which this paper shows has a full potential to do everything. So the cloning itself doesn’t necessarily alter the potential of these ESCs.”
Can Two-Step Side-Step Human ESC Embargo?
BioWorld Today asked Jaenisch whether his method in some respects avoids or obviates the limited list of human embryonic stem cells that President George Bush has authorized for research.
“That’s a pretty complicated question,” he responded. “Many people are opposed to cloning to reproductive cloning mostly which is advocated by some of these cloning activists. I think theirs is a totally irresponsible effort that should be stopped.
“But therapeutic cloning,” he observed, “doesn’t involve making an implanted embryo only an embryonic stem cell line, which never can make a baby. In my opinion, this is less of an ethical problem than making a stem cell from a fertilized embryo. So I think it does impinge on this issue, but doesn’t resolve it. It really raises an ethical, societal and moral question of such complexity that often the public is confused.”