Imaging system helps to advance therapeutic cloning
Embryonic stem cells can contribute to a range of therapies targeting aging or diseased cells. However, the stem cells to which researchers currently have access are derived from in vitro fertilized embryos, and any transplant using them likely would result in rejection unless immunosuppressive drugs were applied indefinitely.
Researchers can overcome these obstacles by producing embryonic stem cells that are genetically identical to the patient’s. Recent studies suggest that such cells can be derived through epigenetic reprogramming of somatic cells -- typically skin cells -- using somatic cell nuclear transfer, essentially converting the cells to stem cells by removing the nucleus and inserting it into an ovum, thus sidestepping the need for fertilization. This technique, more commonly known as therapeutic cloning, has proved successful in mice, but translation to primates has remained problematic.
Researchers have successfully converted skin cells to stem cells in a rhesus monkey, advancing the possibility of therapeutic cloning in humans. They achieved this by removing the meiotic spindles from the eggs of female rhesus monkeys and inserting DNA material obtained from the skin cells of an adult male rhesus monkey.
A team with Oregon Health & Science University in Beaverton, with the Chinese Academy of Sciences in Beijing, and with the University of Nebraska Medical Center in Omaha recently noted a correlation between incomplete nuclear remodeling in the wake of standard somatic cell nuclear transfer in rhesus macaque monkeys and a decline in maturation-promoting factor activity. Following this observation, they developed protocols designed to prevent such a decline.
The system facilitates the imaging of structures that are otherwise virtually invisible, without staining them for fluorescence imaging. The egg shown here is about to be injected with a sperm in a fertility clinic. The bright object near the bottom of the egg is the spindle, which the authors of the Nature paper had to remove from the monkey eggs to achieve therapeutic cloning. A pseudo-color close-up of the spindle, showing some of its internal structure, appears in the upper right corner. Image courtesy of Cambridge Research & Instrumentation Inc. of Cambridge, Mass., and Reproductive Medicine Associates of New Jersey.
In the Nov. 22 issue of Nature, the researchers with Oregon Health & Science University in collaboration with investigators from the University of Nebraska Medical Center and from Whitehead Institute for Biomedical Research in Cambridge, Mass., reported the successful application of the protocols to therapeutic cloning in rhesus macaques, suggesting a translational model with which to verify the feasibility, effectiveness and long-term safety of therapeutic cloning in humans.
The success of the study was due in large part to the Oosight, an imaging system made by Cambridge Research & Instrumentation Inc. of Woburn, Mass., that is an attachment for microscopes based on technology invented and developed at the Marine Biological Laboratory in Woods Hole, Mass. The system allowed the researchers to remove the meiotic spindle, with the DNA material attached, from the eggs of female rhesus monkeys without having to stain the eggs. Such “enucleation” is the first step in therapeutic cloning.
A novel imaging system enabled researchers to visualize the DNA material without resorting to staining, which can damage the cells. The combination of this system and the newly developed protocols for somatic cell nuclear transfer allowed them to derive two viable stem cell lines. Shown here is a colony of cells from one of these lines.
“We wanted to spot the DNA material,” said Shoukhrat M. Mitalipov, the principal investigator of the study. “This imaging system helps you to see the spindle without using any harmful chemicals.”
Development of the technology used for the system began in the 1950s, when Shinya Inoué used a polarized light microscope to visualize the birefringent components of a cell and thus observed, for the first time, meiotic spindle fibers in living cells. In 1957, he achieved a dramatic reduction in the distortion and a much increased contrast by adding a polarization rectifier to the system. The mid-1990s saw further improvement when Rudolf Oldenbourg incorporated liquid crystals with electro-optical controls and software. Thus was born the LC-PolScope, which provides for the simultaneous measurement of birefringence in all resolved specimen points in the microscope’s field of view, as opposed to the single point accessible with traditional microscopes.
Cambridge Research & Instrumentation continues to develop the technology in collaboration with advanced users, including Dr. David L. Keefe of the University of South Florida in Tampa and colleagues at the Marine Biological Laboratory. “One of the key technologies in producing the instrument is the liquid-crystal components that we fabricate,” said the company’s David Fletcher-Holmes. Using this technology, they illuminate the sample with circularly polarized light and then filter the light exiting the sample with a computer-controlled liquid-crystal polarizer that may produce any desired polarization state.
“With these two sets of optics, we can determine exactly what changes in polarization must have been induced by the sample,” he added. “That is, we can fully characterize the birefringence of the sample, enabling us to noninvasively study its molecular structure.”
When these optical measurements are completed, the accompanying software uses proprietary algorithms to generate intuitive imagery and data that allow scientists to see hitherto invisible structures and to quantify their integrity. This can contribute to in vitro fertilization (IVF) as well as to the technique developed by Mitalipov and co-workers. “In the world of IVF,” Fletcher-Holmes said, “embryologists often use the automated reports from our software as a guide to the comparative viability of human eggs, so that the best embryo can be found for re-implanting into the patient.” These automated reports are especially relevant at a time when fertility treatment regulatory bodies the world over are looking to inhibit the practice of boosting success rates by transferring many embryos back to the patient, a practice that can increase the likelihood of a multiple pregnancy.
Using the system, the authors of the Nature paper removed the meiotic spindles from the eggs of 304 female rhesus monkeys and then inserted DNA material acquired from the skin cells of an adult male rhesus monkey. They allowed the cells to grow to the blastocyst stage, from which they obtained two viable stem cell lines that proved to be genetically identical to the adult monkey. Both demonstrated normal stem cell morphology, expressed important stem cell markers and differentiated into a variety of cell types, in vivo as well as in vitro.
Thus, the new approach to enucleation could advance therapeutic cloning to the point where it is feasible in primates, possibly enabling the clinical implementation of the technique. In addition, human stem cells derived with somatic cell nuclear transfer could contribute to in vitro studies designed to probe disease mechanisms and to develop potential treatments.
Further refinement to the technique is needed, though. Mitalipov noted, for example, that the efficiency is still low: 100 to 150 eggs are required to obtain a single cell line. “If we were to apply it to humans, we would have to get that many eggs.” To improve the efficiency, the investigators are studying reprogramming -- the process by which the egg converts the somatic cells to stem cells. They investigate factors in the egg that are involved in this process and are exploring the mechanisms by which it occurs.
“Once we understand these,” he added, “we hope to trace what proteins are actually responsible for reprogramming” and then to improve the efficiency of the technique by adding or overexpressing these proteins.
Contact: Shoukhrat M. Mitalipov, Oregon Health & Science University; e-mail: firstname.lastname@example.org; David Fletcher-Holmes, Cambridge Research & Instrumentation Inc.; e-mail: email@example.com.
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