Then came the “eureka” moment. On that February afternoon, as Hübner peered into her microscope, her eyes met the faint glint of green. Germ cells.

“It was like, ‘Whoa, they’re green green!’” Hübner remembers.

Green as they were, they were scant in number. So few that Hübner wondered whether they really were germ cells. But when she checked in on them the next day, she discovered more cells that had cropped up overnight: “I started photographing like crazy and then I ran to Dr. Schöler’s office and said, ‘I think we’ve got them.’ Then everyone started running over to the scope to have a look.”

Over the next few weeks, Hübner ran tests to verify that what she was seeing were germ cells. Then she optimized her culture formula. The picture that emerged was that of a dynamic, ovary-like microenvironment in a dish, where germ cells could mature into oocytes and, in some cases, embryoid structures.

“We were fascinated to see that the mouse cells were capable of producing oocytes that recruit adjacent cells to surround and nurture eggs so structures similar to the natural follicles can form,” Schöler says. “But even more amazing was that the morphological changes were paralleled by physiological function. Proteins and hormones were expressed in a coordinated fashion pretty similar to that of an estrous cycle. The oocytes could even enter meiosis and eventually develop into embryos.”

Stem-cell pioneer Gearhart views this as an excellent teaching model from which to observe how eggs are made. “We may now be able to study oogenesis in a dish,” he exclaims.

By culture Day 8, the Science paper reports, some 40 percent of the plated cells had turned green, indicating early germ cells. GFP expression declined slightly over the next four days as the cells began to separate from each other, similar to germ-cell migration in vivo. ES cell aggregates appeared to behave like follicles, flanking the presumed germ cells and secreting an estrogen precursor. Evidence of meiosis—reproductive cell division—was present after 16 days in culture. Around Day 26, apparent oocytes that had attained the size range of natural, mature oocytes (50-70 microns) were released from their supporting cells, much as normal eggs are released from their follicles during ovulation.

Perhaps most surprising to Schöler and his colleagues was the emergence, at about Day 43, of structures similar to mouse preimplantation embryos. Schöler believes these embryoid structures are parthenotes, embryos that develop from unfertilized eggs. The team also observed several structures that resembled blastocyst-stage embryos, the source for ES cells.

 

In her modest-sized lab, Hübner surveys a group of plates that sit in two large incubators, and she ponders the demands the cells place on her. “Even if it’s a weekend, the cells don’t know,” she says. “They still get hungry and need to be taken care of. That can get annoying.”

Sometimes Hübner works at the incubator hood for six straight hours, followed by another six at the microscope. Despite such tedium, she loves her work. “To see these events going on in the cell nucleus under a scope is one of the most beautiful things there is,” says Hübner, 46, who is invigorated by the chance to peer in on these units that represent the smallest parts of our bodies. “It’s like getting to know yourself. These cells change all the time, and one realizes when they are happy and when they are not happy.”

Hübner removes a culture plate the size of a hockey puck from one of the incubators. It contains a 26-day culture, and she places it under a souped-up microscope that, she says, cost $130,000. The scope, a Leica, is hooked up to a computer with a monitor that facilitates visualization of the green fluorescence. As she adjusts the wavelength, a band
of hazy green—presumably germ cells—shines under the scope.

The green streak, which flanks a denser cluster of support cells that appear grayish, looks moth-eaten. Cell-to-cell contact is critical for germ-cell maturation, Hübner explains. But, as in the body, germ cells maturing in vitro suffer massive die-offs and the survivors gradually lose their tight adherence. Once the remaining oocyte-like structures in the dish are released from their follicular scaffolding, their pursuit of independence causes them to scatter.

Their moth-eaten appearance—easily confused with a failed culture—is probably responsible for the anonymity that cultured eggs seem to have had thus far, Hübner explains: “Morphologically, it does not look attractive because lots of cells are dying. People have told me, ‘I think I’ve seen these cells in the dish and thrown them away.’”

Were it not for the green GFP marker, Schöler and his colleagues might have done the same. Schöler always knew he would one day culture oocytes. It was a conviction that, as far back as a decade ago, he shared with those who inquired about his plans for the GFP he and Hübner were developing.

“They would say to me, ‘Ah, that will never work,’” Schöler recalls.

Although it seems to have worked, the naysayers have not been silenced. “Now,” he chuckles, “they’re saying, ‘Let’s see if these eggs can be fertilized.’”

These days, Hübner is busy refining her culture method in order to improve the cell yield from the current 20-25 oocytes per plate. Her goal is to generate a continuous supply of germ cells in different phases of maturation. The team can then investigate whether the cells they have cultured are truly eggs and, if so, viable ones. Her plans include differentiating the cultured cells, manipulating the culture conditions, performing chromosomal analyses of supposed oocytes, observing meiotic divisions, and attempting fertilizations and implantations.

Schöler et al. are also considering trying to harvest sperm from ES cells in the dish. However, Schöler says, such attempts will likely be more difficult. The reason his ES cells, though male in origin, generate eggs rather than sperm is that production of the latter requires far more complex signaling. “Cells need to relax in a more perfect way in order to become sperm,” he explains. Hence, oocytes are produced as default products.

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2003 The Pennsylvania Gazette
Last modified 09/02/03

FEATURE: The Most Amazing Cell
By Joan P. Capuzzi Giresi
Illustration by Julia Vakser