WASHINGTON, Dec. 6 – Using newer cloning techniques, including the "gentle squeeze" method described by South Korean researchers who earlier this year reported creating the first cloned human embryonic stem cell line, University of Pittsburgh scientists have taken a significant step toward successful therapeutic cloning of nonhuman primate embryos.
It is the first time researchers have applied methods developed in the Seoul laboratory to nonhuman primate eggs. Resulting cloned embryos progressed to the blastocyst stage, a developmental step in which the embryo resembles a hollow, fluid-filled cavity surrounded by a single layer of cells. Called the inner cell mass, this layer contains embryonic stem cells. Growth of a cloned nonhuman primate egg to the blastocyst stage is farther along the developmental spectrum than ever achieved before, Gerald Schatten, Ph.D., director of the Pittsburgh Development Center at Magee-Womens Research Institute, and his colleagues report.
Calvin Simerly, Ph.D., associate professor of obstetrics, gynecology and reproductive sciences at the University of Pittsburgh School of Medicine and the study's first author, will present the findings on Dec. 6 at Cell Biology 2004, the 44th annual meeting of the American Society for Cell Biology. Scientific sessions are Dec. 4 to 8 at the Washington Convention Center in Washington, D.C.
"We've made improvements by adapting some of the Korean methods and have been able to overcome some of the hurdles we were seeing before," said Dr. Schatten, senior author of the study and professor of obstetrics, gynecology and reproductive sciences and cell biology and physiology in Pitt's School of Medicine. "This is a significant step forward and gives us hope for eventually being able to derive embryonic stem cells through therapeutic cloning."
In therapeutic cloning, limited cell division is induced in an unfertilized egg cell to produce embryonic stem cells. In reproductive cloning, an egg cell with a donor nucleus is transferred into a living surrogate female in an attempt to make a successful pregnancy.
Stem cells are believed to be a key ingredient in the body's self-repair system – blank slates that can develop into multiple cell types such as nerve, blood, bone or muscle. Stem cell-based approaches may hold promise for treating or curing diabetes, Parkinson's disease, amyotrophic lateral sclerosis (ALS), heart disease, stroke, spinal cord injury and genetic diseases. Scientists believe that embryonic stem cells may have the most versatility in potential cell-based treatments, but intensive research continues on both embryonic and adult-derived stem cells.
Dr. Schatten and his colleagues are focusing much of their research on strategies to derive embryonic stem cells from nonhuman primates. Such cells could be used as a template for human embryonic stem cell study, and answer many questions about how embryonic stem cells work and whether they can be used safely and effectively against disease or injury.
The Pittsburgh team also is attempting to clone nonhuman primates as a way to generate better research models for human disease so that studies can obtain more accurate results with fewer animals.
While the current study represents significant progress, many barriers to cloning nonhuman primates remain.
Reporting in the journal Science in April 2003, Drs. Schatten, Simerly and their colleagues described fundamental flaws they observed in nonhuman primate embryonic development despite using the techniques of nuclear transfer that had resulted in successful cloning of Dolly the sheep, mice and other domestic animals. In the 2003 study, researchers found basic molecular obstacles that blocked normal cell development, such as absent or deficient proteins, chaotic mitotic spindle structures and misaligned chromosomes. While cell division superficially seemed normal, chromosomal problems existed within each individual cell.
The most recent study appears to have broken that impasse.
"We've had better development to the blastocyst stage in laboratory culture, which may help us to achieve cloned primate embryonic stem cells," said Dr. Simerly. "There are primate embryonic stem cells now, but no cloned primate embryonic stem cells."
Developing cloned primate stem cells is vitally important to evaluate the preclinical safety and immune-tolerance of stem cell transplantation. The primary medical reason for performing therapeutic cloning is to make stem cells that are genetic – and hopefully, therefore, immune matches of the patient's own cells – so that they are not rejected. Being able to generate primate stem cells by nuclear transfer may soon permit transplant investigations to learn whether stem cells created in this way are truly immune-matched.
In addition to replicating the South Korean method of gently squeezing out the egg's nucleus rather than the traditional practice of removing genetic material with a vacuum needle, Pittsburgh researchers performed nuclear transfer with eggs that had not yet achieved full maturity. Cells prepare to divide by moving through various phases of meiosis before actual division takes place at mitosis.
Eggs were enucleated at late meiosis stage I, when the number of chromosomes begins to be reduced by half to make the resulting egg cell ready for fertilization by a sperm cell, which would provide a similar complement of chromosomes. In other animal cloning work, eggs had been enucleated at metaphase II, part of the second stage of meiosis.
Once the maternal genetic material was removed, it was replaced by donor nuclei from rhesus cumulus and fibroblast cells. Cumulus cells surround the developing egg. Fibroblast cells make up connective tissues.
Reproductive cloning remains elusive, however, and the Pittsburgh team's experience thus far indicates the possibility for successful cloning of primates (and perhaps humans) is even more remote than previously believed. Dr. Schatten's group made 135 cloned monkey embryos and transferred them into 25 surrogate female rhesus macaques. No pregnancies were established.
University of Pittsburgh researchers' success in achieving cloned primate embryo development to the blastocyst stage is a significant advancement over first-generation nuclear transfer techniques that had been used to produce cloned primate embryos. In the past, such cloned embryos generally stopped growing at the 8- to 16-cell stage. Even so, cellular development continues to be somewhat flawed, indicating improper nuclear reprogramming and/or other incompatibilities. Spindle abnormalities, motor deficiencies and other chromosomal anomalies were observed. Embryos created through nuclear transfer appear to be inferior to fertilized ones.
"What this shows is that the Korean method for efficient human somatic cell nuclear transfer is equally effective for nonhuman primates, allowing the further progress toward development of an animal model which parallels human biology," said Dr. Schatten. "This approach does not violate federal or state laws, and allows for preclinical investigations that would not be ethically feasible in humans. Our hope is to help advance the preclinical and fundamental knowledge accurately and swiftly so that perhaps clinical trials on stem cell donations might be responsibly considered within this decade."
The study also further illuminates the science of cloning.
"While it would be very important to be able to develop genetically identical primates for disease research, which is one of our aims, we also are investigating the feasibility of therapeutic cloning of stem cells," said Dr. Simerly. "If we can test these techniques in rhesus monkeys, it would go a long way toward discovering whether it is possible to create immune-matched stem cells."
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