Such tools have been commercialized. Several vendors sell reprogramming systems using retroviruses and lentiviruses. A lentivirus produced by Stemgent and sold by Sigma-Aldrich encodes all four of the classic reprogramming factors (Oct4, Sox2, c-Myc and Klf4) in a single construct, with expression activated by doxycycline2. Millipore also offers polycistronic lentiviral reprogramming kits. Life Technologies sells a reprogramming kit that includes Sendai virus. And constructs for these and other tools are generally available from the nonprofit plasmid repository Addgene. Researchers working with mouse cells can use reprogramming systems that allow remarkable control over the transgenes. Separate teams led by Hochedlinger3 and by Rudolf Jaenisch4 at the Whitehead Institute in Cambridge, Massachusetts have crafted mice in which genes for pluripotency factors are placed under the control of doxycycline and inserted into a genomic locus that is never silenced by methylation and therefore is potentially active in any cell in the mice. These so-called reprogrammable mice allow iPS cells to be generated from any tissue: instead of adding viruses to introduce Scientists recognize the cells when they see them: compact colonies with clear edges, and large nuclei that spread almost to the cell membrane. Successful reprogramming experiments make i n d u c e d p l u r i p o t e n t s t e m (iPS) cel ls that resemble embryonic stem cells in both morphology and behavior. Yet appearance can deceive. “I’ve seen a lot of colonies that look beaut i fu l , but when you test them molecularly and functionally, the colonies are not real, not fully reprogrammed,” says Konrad Hochedlinger, a stem cell biologist at Massachusetts General Hospital. Ultimately, iPS cell lines are assessed with functional tests that require several weeks. For mouse iPS cells, that means generating mouse pups; for human iPS cells, testing for differentiation into all major lineages. These tests are time consuming and expensive, and so researchers need reliable ways to pick the best candidate colonies from a reprogramming experiment, much as an employer will sift through resumes before taking the time to interview job candidates. Surrogate markers of quality are important at several steps: picking which colonies to expand, picking which colony expansions to passage into cell lines, and figuring out which cell lines to submit to the most stringent tests. Picking colonies To make iPS cells, researchers introduce a suite of reprogramming factors into cells, usually by adding genes that encode transcription factors active in pluripotent cells. (A similar approach can be used to make differentiated cells directly; Box 1.) Cells respond to the reprogramming factors by dividing to form colonies, some of which can generate iPS cell lines. For reprogramming to go to completion, a cell must restart its own pluripotency genes and silence any introduced genes. Often ‘partially reprogrammed’ cells far outnumber fully reprogrammed ones. These partially reprogrammed cells can closely resemble embryonic stem cell colonies, but they cannot differentiate into the full range of cell types, and their gene expression may be quite different from that of embryonic stem cells. One way to reduce contamination by partially reprogrammed cells is to halt their growth early. For this, researchers sometimes use reprogramming factors that can be turned on and off at will. The introduced pluripotency genes can be constructed so that they are only expressed in the presence of a small molecule like doxycycline; removing doxycycline from the culture medium will silence them. Fully reprogrammed cells have already activated their own pluripotency genes and so continue to grow, but partially reprogrammed cells do not. Non-integrating systems, such as Sendai viruses and episomes, offer a similar advantage. Because they are not copied when cells divide, they are diluted out when cells expand, depriving partially reprogrammed cells of pluripotency factors. Growing cells under feeder-free conditions and with fully defined media can also cut down on the growth of partially reprogrammed cells1. Chimeric mice with all four reprogramming factors in the same genomic locus make producing iPS cells more straightforward. Re f. 3 Konrad Hochedlinger at Massachusetts General Hospital says that many beautiful colonies in reprogramming experiments fail to produce high-quality pluripotent stem cells.