The care and feeding of stem cells

What do embryonic stem cell facilities and intensive-care units have in common?

It’s 2:00 on a Sunday afternoon, and the stem cells are hungry.

Maya Mitalipova, director of the Institute’s Stem Cell Facility, drops whatever she’s doing, exits her Cambridge apartment, and heads over to Whitehead. She hurries to the refrigerator in her lab.

And there they are, thousands of them, clustered on Petri dishes in tiny groups of a few hundred.

Gently, she takes out and sets down the dishes, opens up a second fridge and removes a vial containing the occupants’ favorite food: a formula whose primary ingredient is calf blood. They love the stuff. She warms the vial in a water bath for 15 minutes. Then, pipette in hand, she fills the dishes.

“If I do anything different in their feeding schedule, I may lose 90 percent of a colony,” says Mitalipova. “I get a dramatic reaction if I ignore any aspect of them.”

Mitalipova speaks from years of experience. Before coming to Whitehead, she was already established as a leading expert in culturing and maintaining stem cells at the University of Georgia. And before that, she had isolated stem cells that are now part of the so-called presidential lines.

While maintaining embryonic stem cells may feel a lot like running an intensivecare unit, the cell itself is no more a “patient” than a particle of skin. A stem cell, after all, is just a cell: a membrane and a nucleus buffered by cytoplasm.

But while a skin cell is robust and can live happily on a growth medium with minimal attention, stem cells require an exhausting degree of care giving.

The reason is simple: a skin cell has completed its developmental journey. It can never be anything but a skin cell. It will divide and replicate itself only when it needs to. Otherwise, it simply sits back and drapes your bones.

An embryonic stem cell, on the other hand, is at the starting gate of development. It is pure potential. It hasn’t been assigned a particular fate yet, but it’s dying to get to work and become that liver or brain or hair follicle—anything but a stem cell.

For scientific projects, though, these cells are only valuable to the degree that they are kept from differentiating.

Here’s the dilemma for people in Mitalipova’s position: How do you give an embryonic stem cell everything it needs to thrive, yet keep it from doing the very thing it wants most of all to do?

According to Mitalipova, with great difficulty.

“Timing is critical,” she says. “Neglecting them for just one day can have dire consequences.”

Yes, this is as onerous as it sounds. Seven days a week, someone must attend to the cells. If both Mitalipova and her technical assistant, Ping Xu, need to be away for a few days, they must freeze the cells—an option which is the absolute last resort. “Once you freeze the stem cells, they take two weeks to thaw,” she says.

Checking their IDs

In addition to the daily feedings, Mitalipova needs to continually propagate the stem cell lines so that other researchers in the Institute can use them.

When stem cells are placed in the Petri dish, they immediately divide and start forming clusters, or colonies. For the first few days, this is exactly what you would want.

But once day four or five approaches, it’s time to start getting nervous. “Around this time, each colony has about 500 cells,” says Mitalipova. “Any day each cell will start signaling its nucleus saying, ‘I’m ready to go!’ and it will start trying to develop into some other kind of cell.”

She can tell this is happening simply by taking a good look. Stem cells are perfectly round with dome-like surfaces. When they differentiate they become irregular, less like a spherical drop of water and more like an ink blot.

For reasons that aren’t yet clear, the size of the colony, more than anything else, determines whether or not this happens. When a colony reaches the 500-cell threshold, Mitalipova performs a technique called “passaging.” Here, she adds an enzyme that loosens the cells from their feeder bed, and then she breaks the colony apart into groups of anywhere from 10 to 100 cells. Each of these smaller groups then forms its own colony that will, in about four days, reach the 500 mark, when she will then need to repeat the process. And so on.

“It’s important that I don’t separate them to less than 10 cells,” she says. “Unlike mouse embryonic stem cells, the human cells need cell-to-cell contact in order to survive.”

Life for Mitalipova won’t be getting easier any time soon. Seventeen new lines of human embryonic stem cells recently arrived at the Institute.

“We need to make millions of clones of all the different lines and freeze them. We’ll be feeding them constantly, examining the colonies, measuring their genetic profiles.”

She sighs.

“I won’t have a single day off for the next five months.”