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, Pitt 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 a 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, report Pitt’s Gerald Schatten, senior author of the study, and his colleagues. Schatten is professor and vice chair of the Pitt School of Medicine’s Department of Obstetrics, Gynecology, and Reproductive Sciences and a professor in the school’s Department of Cell Biology and Physiology.

Calvin Simerly, associate professor of obstetrics, gynecology, and reproductive sciences in Pitt’s medical school and the study’s first author, was scheduled to present the findings today (Dec. 6) at Cell Biology 2004, the 44th annual meeting of the American Society for Cell Biology, in Washington, D.C.

“We’ve made improvements by adapting some of the South Korean methods and have been able to overcome some of the hurdles we were seeing before,” said Schatten, who also directs the Pittsburgh Development Center at Magee-Womens Research Institute. “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, in contrast, 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 such multiple cell types as nerve, blood, bone, or muscle. Stem cell-based approaches may hold promise for treating or curing diabetes, Parkinson’s disease, amyotrophic lateral sclerosis, 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.

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, 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 their 2003 study, researchers found basic molecular obstacles that blocked normal cell development, including absent or deficient proteins 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,” Simerly said. “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.

Reproductive cloning remains elusive, 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. Schatten’s group made 135 cloned monkey embryos and transferred them into 25 surrogate female rhesus macaques. No pregnancies resulted.

Pitt 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 eight- to 16-cell stage. Even so, cellular development continues to be flawed, indicating improper nuclear reprogramming and/or other incompatibilities. 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,” Schatten said. “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.”

Pitt’s 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 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.”

A paper describing these findings in more detail will be published in the Dec. 11 issue of the journal Developmental Biology.