ES Cell Culture

Yet the potential rewards of success—from autologous organ transplantation to a cure for diabetes, toxicity testing and protein production, not to mention the wealth of understanding that basic research itself may bring—are promise enough to lure ever more scientists and research dollars into the field. National and international consortia have formed to standardize definitions, protocols, and standards. Eponymous stem cell research institutions, centers, and departments have been founded and endowed. A national hES cell repository has been established. Private companies are being spun off to exploit academics’ findings. And new and established vendors are marketing a gamut of supplies and reagents to support the burgeoning industry.

Nonetheless, a relatively new and unsettled field leaves a lot of open questions, even on the basics. And for those scientists just entering the field, it might be a challenge just to get started, and to know where to go from there.

Stem cells are pluripotent, self-renewing, and (according to some definitions) capable of functionally reconstituting a given tissue in vivo. ES cells—derived from the blastocyst of mouse, human, and several other species—fulfill at least the first two of those three criteria. Several recently described cell types—such as the multipotent adult progenitor cells (MAPC) and marrow-isolated adult multilineage inducible (MIAMI) cells—may do so as well, although their pluripotency has yet to be verified in vivo. And, notes James Battey, chair of the NIH Stem Cell Task Force, in a recent webcast, ES cells are the only non-malignant cells currently known to have the capacity for unlimited self-renewal.

If you’re investigating how cells retain their plasticity (that is, the ability to differentiate into different cells) or the signaling pathways involved in the early stages of differentiation, or if you’re looking into banking cells as a safeguard against any future diseases, then ES cells are perhaps the only ones suitable to the task, given current technology.

However, if your research is on a particular cell-type—a blood precursor, for example, or a neuron—or tissue type, then you may want to consider beginning with more differentiated, but still multipotent, precursors, such as hematopoietic or neuronal stem cells. Besides side-stepping the ethical minefield of obtaining human embryo-derived cells, “adult stem (AS) cells” have some advantages over their more plastic cousins. For example, notes Peter Quesenberry, director of the Cancer Center at the Roger Williams Medical Center in Providence, ES cells tend to form tumors and cancers when transplanted into a mouse, yet “adult stem cells appear to work quite well.”

It’s also difficult to keep 100% of an ES culture undifferentiated, points out Eric Bouhassira, director of the Einstein Center for Human Embryonic Stem Cell Research at the Albert Einstein College of Medicine. “Every paper that does profiling using microarrays describes how they try to get pure ES cell populations,” he says, “but when you compare the results among the different papers, they’re not so concordant.”

Mouse ES cells are easier to work with, have fewer ethical, safety, and regulatory considerations and hurdles to deal with, and are better characterized. There are far more reagents available for mouse lines. Mouse ES cells also have a far better freeze/thaw survival rate. And new lineages can easily be derived (and funded), allowing the role of isolation and early propagation issues to be explored.

Yet there are some major differences—significant enough that what you learn on one may not directly translate to the other.

For example, while hES cells can be induced to make extra-embryonic tissues such as trophoblast, chorion, and placenta, mES cells thus far can only differentiate into embryonic structures, notes Jonathan Auerbach, director of the Stem Cell Center at the American Type Culture Collection (ATCC).

Some of the factors required to maintain ES cell culture differ among species as well. Leukemia Inhibitory Factor (LIF), for example, was identified as one of the MEF-derived molecules required to maintain self-renewal in mES cells, but appears to play no role for hES cells.

While academic laboratories until very recently had to pay $5000 per NIH-registered hES cell line, as of November 2005 ATCC charges $650, and the WiCell Research Institute is promising to drop the cost for the lines it sells to $500—comparable to the cost of mES cells. Still, Auerbach recommends that researchers purchase and learn to culture karyotypically abnormal hES lines before investing in the real thing. “Those are a little bit easier to grow because they’ve gone through this adaptation phase in culture,” he says. “We like to call them ES cells with training wheels: the morphology is the same, and the characterization would be the same.”