Pluripotency for the Masses

The cells themselves

User: Mark Ungrin, postdoc in Peter Zandstra's lab at the University of Toronto

Project: Improving consistency and yield in hESC differentiation by creating aggregates of hESCs from single cell suspensions.

Problem: Techniques for differentiating hESCs and hiPSCs require the formation of cell aggregates. But the clumps are usually heterogeneous in size and shape, leading to an inconsistent differentiation yield. To achieve consistency Ungrin tried a published protocol for creating aggregates from single cell suspensions. Initially, he was able to create stable aggregates but then they stopped surviving. "The aggregate size just wasn't consistent from experiment to experiment," he recalls. "Sometimes I'd get multiple aggregates within a single well."

Solution: Ungrin spent six months troubleshooting the culture media to no avail. Then, he turned to the cells themselves, finding that two factors seemed to help: To his surprise, older hESCs, which had been passaged many times, were more likely to form stable aggregates. And cells in the predifferentiation stage seem more likely to survive reduction to single-cell suspension, so Ungrin made sure to harvest the population before all of the cells had differentiated. He also found that a small molecule inhibitor of Rho-associated kinase, shown in previously published studies to promote survival, worked well with his cells (PLoS ONE, 3:e1565, 2008).

Considerations: "One of the bugbears of working with human ES cells, is that things that you do in one lab can be difficult to reproduce for reasons that are not obvious," he says. Even differences between how two different lab technicians pipette the cells from one plate to another can affect survival. When troubleshooting, Ungrin suggests remembering that the age and state of your cells, in addition to the culture media, can affect survival.

Marker manhunt

User: Raj Rao, stem cell bioengineer at Virginia Commonwealth University, Richmond

Project: Characterizing and monitoring human embryonic stem cells using cell surface markers.

Problem: There are no set standards for assessing the pluripotency of hESCs and how the cells change as they differentiate. Commonly used cell surface markers include stage-specific embryonic antigen-3 and 4 (SSEA-3 and SSEA-4). But different cell lines vary in their expression of SSEA-3 and SSEA-4. "It makes sense to have a battery of markers," says Rao. So he set out to find more.

Solution: Rao chose to use lectins, proteins that bind and modify cell surface sugars throughout embryonic development. According to the literature on mouse ESC characterization, lectin receptors are displayed on cell surfaces at preimplantation and implantation stages of development. The group tested 14 different lectins, looking for ones expressed with SSEA-4. They pulled out a few lectins, such as Ricinus Communis agglutinin and Maackia amurensis, that bound tightly to the hESCs, predicting pluripotency.

Considerations: The more markers you test, the more certain you can be about the pluripotency of your cells, Rao says. For those who want to try characterizing based on lectins, it's a relatively straightforward and inexpensive process; you'll need a cohort of antibodies and access to a table-top flow cytometer. Remember though, expression of lectin and other cell surface markers such as SSEA-4 varies between cell lines and even colonies from the same line. One way to increase your confidence in the results is to increase the number of samples you analyze.

Mycoplasma mania

User: Travis Berggren, stem cell core facility director, Salk Institute for Biological Studies, La Jolla, Calif.

Project: Studying the mechanisms controlling hESC self-renewal mediated by fibroblast feeder layers.

Problem: When Berggren worked as a staff scientist at the WiCell Institute in 2005, some of his hESCs started detaching from their cell culture dishes and dying. Many of the cells in the lab were contaminated with mycoplasma, a type of bacteria that is antibiotic resistant and typically not visible.

Solution: The group spent two weeks purging their cells and culture reagents, autoclaving every incubator, and bleaching and testing the facility. But they lost more than just two weeks of work. Since mycoplasma starts growing slowly and doesn't initially interfere with differentiation, it's likely the researchers unknowingly worked with contaminated cells for longer than a month. Even after detecting mycoplasma, some coworkers still thought their cells looked viable, even though they tested positive, Berggren says. "That tells you that you really need to test early on."

Berggren's group established a testing routine which they still follow religiously three years later. They test new cultures weekly and older cultures every 2-4 weeks, by testing for DNA or enzymes specific to mycoplasma. All new culture materials, such as mouse fibroblasts used in the feeder layer, are put in a quarantined incubator. They don't enter the lab's main stream of culture materials until they've tested clean at least twice.

Considerations: Mycoplasma is especially troublesome for labs that move from work in mouse ESCs to work in human ESCs because human stem cells suffer much more severe consequences as a result of exposure to it, by losing their full differentiation potential and/or dying. Labs working with hiPSCs must also watch out that the cells used to deliver the pluripotency factors are not contaminated.

Dynamics of differentiation

User: Wu Ma, developmental biologist, ATCC Stem Cell Center, Manassas, Va.

Project: Maintaining hESCs for later conversion into neural progenitor cells.

Problem: Ma knew the extra-cellular matrix (ECM) was critical for stem cell renewal and differentiation, but he wanted to determine which elements of the ECM helped stem cells form neural progenitor cells.

Solution: Ma's group tested two different hESC lines, plating each onto five different surfaces: poly-D-Lysine, fibronectin, laminin, type 1 collagen, and Matrigel (an ECM product sold by BD Biosciences). He found that cells cultured in laminin were best at differentiating into neurons. That effect was partially mediated by the stem cells's integrin receptor: blocking the a6b1 receptor slowed differentiation.

Considerations: Laminin worked best for Ma, but there's no one-size-fits-all answer to which medium to choose. Different culture media are constantly being developed and the efficacy of one culture can even vary from cell line to cell line, Ma says.

Ma advises when choosing culture media and other materials, to read up not only on how past studies stem cell studies have addressed the issue of microenvironment, but also on factors known to be involved in normal in-vivo development. If you're differentiating to neurons, for example, familiarize yourself with the literature on factors involved in brain development. But know the limits of the literature, says Ma. "Under the same culture condition some human ESC lines produce more neurons than others" because each line has its own genetic background.