Working with embryonic stem cells from mice, researchers at Children's Hospital Boston/Harvard Medical School and the Whitehead Institute for Biomedical Research created a continuously growing line of embryonic germ cells - primitive cells in the embryo that mature to become sperm or eggs -- providing for the first time an opportunity to study this process in the Petri dish. In a second step, they created male gametes that were capable of fertilizing an egg to form an early embryo. The research is published in the Dec. 10 online edition of the journal Nature.
EMBRYONIC GERM CELLS
Embryonic germ cells are a unique group of cells that the embryo sets aside for future reproduction. An early embryo starts with only about 50 of these rare cells, making them very difficult to isolate and study. Using embryonic stem cells from mice, the researchers first created multi-cellular structures called embryoid bodies that have some characteristics of early mouse embryos and the beginnings of differentiated tissue (muscle, blood, etc.). The embryoid bodies were allowed to grow for 4-10 days.
Next, the researchers isolated germ cells from the embryoid bodies and cultured them in a dish along with growth factors. The result was a continuously growing line of embryonic germ cells. These methods now provide a cell culture system in which the unique properties of embryonic germ cells can now be more easily studied.
Parental imprints and cancer
First, the researchers want to study embryonic germ cells to better understand a set of genetic instructions called imprints. Imprints affect a small group of about 50 genes, many of which govern growth, and determine which copy of the gene is turned on - that coming from the mother, or that from the father. Cells maintain these imprints throughout life - but in embryonic germ cells, the imprints are erased.
This erasure has direct implications for cancer research, says Niels Geijsen, Ph.D., lead author of the study and now a principal investigator at the Center for Regenerative Medicine and Technology at Massachusetts General Hospital. In certain tumors, he explains, imprints that tend to slow down growth and cell division have been lost, so cells grow out of control. "We hope to use these embryonic germ cells to study how the erasure process is initiated normally, and how it is disrupted in some cancers," he says.
Studying the imprints may also offer insights into the process of cloning, the researchers note. The cloning process, which involves transferring the nucleus of an adult cell into an egg, causes many of the parent-specific imprints to be lost. This loss is responsible for many of the abnormalities found in cloned animals.
Embryonic germ cells may also aid in understanding the process of cell specialization. Most of an embryo's cells - and all adult cells - are programmed to specialize and create particular tissues and body parts via a separate set of gene modifications. But embryonic germ cells are the only cells within the developing organism to maintain the capacity to generate all tissues, also called pluripotency.
"We want to know how these cells are kept in their pristine state," says George Daley, M.D., Ph.D., senior investigator on the study and a stem cell biologist now at Children's Hospital Boston and Harvard Medical School. "This may teach us about how we can reverse the process and reprogram adult cells back to their embryonic state."
A better understanding of how these gene modifications are programmed may come from studying the parent-specific imprints, and may have implications for therapeutic cloning, Daley adds. In therapeutic cloning, scientists take a cell from an adult animal, such as a skin or blood cell, remove its nucleus, which contains all the genes, and place that nucleus inside an egg whose own nucleus has been removed. This process of nuclear transfer creates an early embryo and somehow - no one knows quite how - erases the genetic markers instructing the cell to differentiate. If scientists could learn how the erasure happens, it might be possible to create stem cells capable of generating all tissues without having to obtain a donor egg for cloning, and without having to create a new embryo - in fact, without having to clone at all.
In the second phase of the research, the investigators asked whether the germ cells could be coaxed into becoming functional gametes - reproductive cells -- under laboratory conditions.
Earlier this year, Hans Schöler and colleagues from the University of Pennsylvania reported being able to make female gametes -- eggs -- from mouse embryonic stem cells. However, these eggs so far have not proven capable of being fertilized by sperm. Daley's team worked on the other side of the reproductive equation, creating male gametes, or primitive male sperm.
The researchers used the same embryoid bodies that generated the embryonic germ cells, but this time, they grew the bodies for two to three weeks, allowing the germ cells to mature into male gametes that could be isolated in the lab. These gametes were not full-fledged sperm - for instance, they had no tails. However, when Kitai Kim, Ph.D., a postdoctoral fellow in Hematology/Oncology at Children's Hospital, and Kevin Eggan, a Junior Fellow in the Harvard Society of Fellow, injected the gametes into eggs, the gametes fertilized the eggs and fused their DNA with that of the eggs, creating embryos with full sets of chromosomes.
The researchers now plan to take the next step, transferring these early embryos into female mice to see if they develop. "We will see if they make pups," Geijsen says. "This will tell us how normal the gamete really is."
Infertility, birth defects
The ability to make both embryonic germ cells and male gametes in the lab is unprecedented and has powerful implications for understanding how germ cells mature, and may suggest new approaches to infertility, says Geijsen. "A lot of problems with infertility start very early," he says. "Something goes wrong in the germ cell at a very early stage."
Geijsen wonders, for example, why they were able to create far more embryonic germ cells than gametes in the lab - what made the germ cells stop developing? The ability to get more of the germ cells to mature into functional gametes may someday help infertile men who cannot make full-fledged sperm that would allow them to father children, but do have primitive sperm stem cells.
Being able to study the very earliest stages of development may also shed light on birth defects, Daley adds. "Germ cells are given the responsibility for perpetuating the species, and understanding how germ-cell formation goes awry may teach us about early developmental defects, as well as some forms of male infertility," he says. "Our research is aimed at understanding normal and pathologic tissue formation, and not so much at futuristic means of assisted reproduction."
The research was funded by the National Institutes of Health, the National Science Foundation, and the Dutch Cancer Society. Individuals on the research team received support from the Leukemia and Lymphoma Society and the Harvard Society of Fellows.
Children's Hospital Boston is home to the world's largest research enterprise based at a pediatric medical center, where its discoveries have benefited both children and adults for more than 130 years. More than 500 scientists, including seven members of the National Academy of Sciences, nine members of the Institute of Medicine and nine members of the Howard Hughes Medical Institute comprise Children's research community. Founded in 1869 as a 20-bed hospital for children, Children's Hospital Boston today is a 300-bed comprehensive center for pediatric and adolescent health care grounded in the values of excellence in patient care and sensitivity to the complex needs and diversity of children and families. It is also the primary pediatric teaching affiliate of Harvard Medical School. For more information about the hospital visit: www.childrenshospital.org.