9 October 2008. Less than a month before Americans elect a new president—a decision with potential science policy ramifications—perhaps it’s fitting to see a recent flurry of papers offering safer, less controversial methods for generating host-specific stem cells. Capable of differentiating into cells that form any tissue in the human body, these rare precursors have long lured researchers who study degenerative disease, including Alzheimer’s. Two weeks ago, Boston scientists described an alternative protocol for generating induced pluripotent stem (iPS) cells in mice using non-integrating viral vectors with reduced risk of tumorigenicity in host cells (see ARF related news story). Now, in a report appearing today in Science online, the Japanese group credited with the original version of that procedure has gone a step further by eliminating viral vectors altogether. Meanwhile, researchers in Germany reported yesterday in Nature online a first-ever method for producing human pluripotent stem cells from testicular biopsies.
During development, embryonic stem (ES) cells become progressively committed to specialized cell lineages—a stepwise process that is typically irreversible. Over the years, though, scientists have come up with molecular tricks to send adult somatic cells back toward infancy such that they assume developmentally less committed states with the potential to form a variety of new cells. The introduction of four transcription factors (Oct4, Sox2, c-Myc, and Klf4), in fact, is all it takes to get mouse fibroblasts to revert to an embryonic-like state (Takahashi and Yamanaka, 2006). This protocol—initially published by Shinya Yamanaka of Kyoto University, Japan, and colleagues—has drawn wide praise but carries a major safety concern with its use of viral vectors that can integrate into the host genome and cause tumors. In the new study, Yamanaka’s group was able to generate iPS cells from mouse embryonic fibroblasts (MEFs) using a method completely devoid of viruses. Instead, the reprogramming factors finagled their way into host fibroblasts on plasmids. The host cells came from mice in which green fluorescent protein (GFP) and the puromycin resistance gene are driven by regulatory elements of the ES cell gene Nanog (Okita et al., 2007). In this system, iPS cells appeared as puromycin-resistant, GFP-positive colonies.
After toying with different vectors and gene arrangements, first author Keisuke Okita and colleagues found success with a two-plasmid, serial transfection procedure. On days 1, 3, 4, and 7, they transfected Nanog-reporter MEFs with two plasmids—one containing Oct3/4, Klf4, and Sox2, and another carrying c-Myc. The resultant GFP-positive colonies produced cells that resembled ES cells in terms of morphology and gene expression. Most likely, the iPS cells did not contain integrated DNA from transfected plasmids, as PCR (using 16 primer sets) and Southern blotting failed to detect plasmid DNA amplification in nine of 11 GFP-positive clones. To confirm pluripotency of the iPS cells, the researchers transplanted them subcutaneously into nude mice (which lack a thymus and hence cannot mount T cell-mediated graft rejection responses). All 10 tested iPS clones produced teratomas containing representative cell types of the three germ layers and, when injected into blastocysts, gave rise to chimeras with no PCR-detectable transgene integration. (Teratomas are multilayered, benign tumors that serve as a landmark test for the developmental potential of iPS cells—not to be confused with the lethal tumors resulting from integration of viral vectors into host genome.) Disappointingly, though, one million transfected cells gave rise to just one to 29 GFP-positive colonies—an efficiency 10- to 100-fold lower than seen with the original protocol using retroviruses. In this regard, the new plasmid-based procedure was quite comparable to the recently reported protocol using non-integrating adenovirus (see ARF related news story). However, that method—in which 500,000 transfected cells produced three ES-like colonies—used host cells from adult mouse liver, whereas the new study was done in embryonic fibroblasts.
Approaching the challenge of stem cell generation from a different angle, a team of German researchers reported the first successful derivation of pluripotent stem cells from adult human testis. Led by Arnulf Stenzl, University Clinic Tuebingen, and Thomas Skutella, Center for Regenerative Biology and Medicine, also in Tuebingen, the researchers extend similar lines of studies in mice using neonatal (Kanatsu-Shinohara et al., 2004) and adult (Guan et al., 2006) testis, and germline progenitor cells (Seandel et al., 2007).
Starting with functional tissue from 22 human testicular biopsy samples, lead author Sabine Conrad and colleagues cultured single cells for four days with glia-derived neurotrophic factor (GDNF) to spur self-renewing division of spermatogonial stem cells. To enrich for this rare population and deplete somatic cells, the researchers used magnetic-activated cell separation (MACS) with CD49f (α6 integrin) followed by a matrix selection procedure with collagen and laminin. Further culture and differentiation of the resultant cells yielded pluripotent germline stem cell (GSC) clusters 21 to 28 days later. The GSC cells appeared very similar to human ES cells by a host of cellular and molecular features revealed by immunostaining, fluorescence-activated cell sorting, reverse-transcription PCR, Western blot, and microarray analysis. Importantly, GSC cells made from eight different patients produced characteristic teratomas when transplanted into immunodeficient mice and differentiated in vitro into various somatic cell types of all three germ layers. The authors say the method may provide “simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.”—Esther Landhuis.
Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S. Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors. Science. Abstract
Conrad S, Renninger M, Hennenlotter J, Wiesner T, Just L, Bonin M, Aicher W, Buehring H-J, Mattheus U, Mack A, Wagner H-J, Minger S, Matzkies M, Reppel M, Hescheler J, Sievert K-D, Stenzl A, Skutella T. Generation of pluripotent stem cells from adult human testis. Nature. Abstract