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.

Reproductive Cloning Remains Elusive

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.

Cellular Development Flawed

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.”